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Home , Antiserum, Polyclonal antibodies

Canadian Council on Animal Care

guidelines on: production This document, the CCAC guidelines on: antibody production, has been developed by the ad hoc subcommittee on immunological procedures of the Canadian Council on Animal Care (CCAC) Guidelines Committee:

Dr Albert Clark, Queen's University (Chair) Dr Dean Befus, University of Alberta (Canadian Society for representative) Dr Pamela O'Hashi, University of Toronto Dr Fred Hart, Aventis Pasteur Dr Michael Schunk, Aventis Pasteur Dr Andrew Fletch, McMaster University Dr Gilly Griffin, Canadian Council on Animal Care

In addition, the CCAC is grateful to the many individuals, organizations and associations that pro- vided comments on earlier drafts of this guidelines' document. In particular thanks are extended to: the Canadian Society of Immunology; the Canadian Association for Laboratory Animal Science and the Canadian Association for Laboratory Animal Medicine; Dr Terry Pearson, University of Victoria; Dr Mavanur Suresh, University of Alberta; Drs Ernest Olfert and Barry Ziola, University of Saskatchewan; and Dr Patricia Shewen, University of Guelph.

© Canadian Council on Animal Care, 2002

ISBN: 0-919087-37-X

Canadian Council on Animal Care 315-350 Albert Street Ottawa ON CANADA K1R 1B1

http://www.ccac.ca CCAC guidelines on: antibody production, 2002

TABLE OF CONTENTS

A. PREFACE ...... 1 E. REFERENCES ...... 20

B. INTRODUCTION ...... 2 1. General ...... 20 2. Polyclonal Antibody Production ...... 21 C. POLYCLONAL ANTIBODY PRODUCTION 3 3. Production . . . . .25 3.1 Other useful references ...... 27 1. Animal Selection and Care ...... 3 3.2 Additional useful information . . . . .27 2. Immunization Protocol ...... 6 3. Standard Operating Procedures ...... 6 F. GLOSSARY ...... 27 4. Preparation ...... 6 5. Choice of Adjuvant ...... 7 6. Route of Injection ...... 9 7. Volume and Number of Injection APPENDIX A Sites ...... 10 8. Blood Collection ...... 12 COMMON ADJUVANTS ...... 31 9. Monitoring of Animals ...... 13 10. Disposition of the Animals ...... 13 APPENDIX B

IMMUNIZATION – RECOMMENDED D. MONOCLONAL ANTIBODY ROUTES AND VOLUMES PRODUCTION ...... 14 (adapted from Leenars, et al., 1999) ...... 33

1. Animal Selection and Care ...... 16 2. Production of Hybridoma Clones . . . . .17 APPENDIX C 3. Ascites Production ...... 17 STAGES OF MONOCLONAL 3.1 Priming ...... 17 ANTIBODY PRODUCTION ...... 35 3.2 Contamination ...... 18 3.3 Hybridoma implantation ...... 18 APPENDIX D 3.4 Monitoring the animals and endpoints ...... 19 INFORMATION ON IN VITRO 3.5 Ascites tumor growth ...... 19 TECHNIQUES FOR MONOCLONAL 3.6 Ascites fluid collection ...... 20 ANTIBODY PRODUCTION ...... 37 antibody production

A. PREFACE on antibody production given in the 1991 CCAC policy statement on Acceptable The Canadian Council on Animal Care Immunological Procedures. (CCAC) is responsible for overseeing animal use in research, teaching and test- Many institutions already have excellent ing. In addition to the Guide to the Care and standard operating procedures (SOPs) in Use of Experimental Animals,Vol. 1, 2nd place for the production of both pAbs and Edn., 1993 and Vol. 2, 1984, which lay mAbs. These CCAC guidelines borrow down general principles for the care and much from the experience of these institu- use of animals, the CCAC also publishes tions, and from the recent international guidelines on issues of current and emerg- initiatives to refine protocols for antibody ing concerns. The CCAC guidelines on: production. antibody production is the fifth of this series, and has been developed by the CCAC ad The refinement of animal use in research, hoc subcommittee on immunological proce- teaching and testing is an ongoing process dures. which is never complete. The CCAC recog- nizes that in moving towards implementa- The purpose of this document is to present tion of these guidelines, considerable guidelines for production of both polyclonal expenditure may be involved; for example, (pAb) and monoclonal (mAb) that to establish central services for production assist investigators and research support of mAbs in vitro. As with the implementation personnel to achieve an acceptable of other CCAC guidelines, institutions must immunological result with minimal discom- recognize such expenditures as responsi- fort for the animals involved. These guide- bilities if sound humane research is to be lines are also provided to assist animal carried out within their facilities. The CCAC care committee (ACC) members to evaluate is committed to assisting institutions by protocols involving the production of providing information on production of mAbs antibodies, to ensure that the highest in vitro as well as on best practices for standards of animal care and use are met. animal-based production for pAbs and These guidelines supercede the guidance mAbs where necessary. B. INTRODUCTION and from a single animal over time. There- fore, pAbs have a finite availability and are Antibodies are produced by the immune subject to possible character change during system of an animal in a specific response the period of production. On the other hand, to a challenge by an immunogen. The monoclonal antibodies (mAbs) are derived acts through two principal from a single and hence are specific mechanisms: humoral type responses for a single and have a defined (production of antibodies) and cell-mediated affinity for that epitope. Thus if the right responses. () are mAb is obtained, it can be extremely speci- molecules which can induce a specific fic for the relevant immunogen, and under and are usually foreign appropriate conditions, an almost limitless production of a constant product is possible. ccac guidelines proteins or carbohydrates, or sometimes lipids and nucleic acids. The immune systems of mammals are comprised Polyclonal antisera can be obtained in a of large numbers of , each relatively short time frame (1-2 months), in characterized by its unique -receptor contrast to standard mAb production specificity. This receptor diversity permits procedures that can be tedious and require immune responses to a broad range of 3-6 months. (Newer procedures, however, immunogens. The B lymphocytes, charac- can shorten the time for mAb derivation to terized by the presence of specific immuno- as little as one month.) Polyclonal anti- globulin receptors on their surface, are bodies show different affinities for different responsible for production of the humoral and thus may demonstrate overall (antibody) response. Antibodies are secret- excellent binding achieved by adherence to ed from plasma cells which have differen- a number of different sites on a complex tiated from B lymphocytes after appropriate immunogen or antigen. In contrast the stimulation by the foreign immunogen. single epitope specificity of mAbs may Each antibody molecule is capable of mean that a slight change in the structure of recognizing a specific epitope (antigenic the epitope, for example by antigen denatu- site), usually 5-6 amino acids or monosac- ration (such as occurs in immunoblotting charide units which are either linear or experiments or by sterilization of the topographically assembled, and is capable immunogen prior to injection into the of binding to this epitope. animal), can result in loss of antibody binding. For this reason two or three mAbs A polyclonal humoral response is comprised are sometimes combined. of antibodies, derived from various clonal populations having varying specificities (for Consideration should be given at the outset different epitopes, even on the same mole- to procuring commercially produced anti- cule), affinities and classes, and hence bodies from sources that comply with CCAC provides an effective defense against guidelines, or if international, are accredited pathogens. Polyclonal antisera are difficult by the Association for Assessment and to reproduce because of the variety of Accreditation of Laboratory Animal Care, antibodies made in the polyclonal response. International (AAALAC). The Antibody The level and quality of the antibodies Resource Site http://www.antibodyresource. produced will vary from animal to animal, com may assist in this regard as well as

2 provide background information on antibody immunogens or antigens (Hanly, et al., production. A list of suppliers of mAbs 1995). A number of references to suitable produced in vitro has been compiled by the immunology texts are given in the reference Focus on Alternatives Group, UK, and a list: Abbas, et al., 2001; Anderson, 1999; second list of US suppliers has been com- Kuby, 2000; Roitt, et al., 1998; Sharon, piled by the Alternatives Research and 1998; Sompayrac, 1999.

Development Foundation, US. Both lists antibody production, 2002 can be found at http://www.frame.org.uk/ General guideline: In the production of Monoclonal_Suppliers.htm. Additional , the overriding material, for example SOPs, are available consideration must be to minimize pain on the CCAC website http://www.ccac.ca. and distress for the animals used.

1. Animal Selection and Care C. POLYCLONAL ANTIBODY PRODUCTION Guideline: Careful consideration should be given to the selection of the species Polyclonal antibodies (pAbs; antisera) have to be used for polyclonal antibody a number of uses in research, for example: production. for detection of molecules in ELISA-type assays; in Western blots; in immuno- Careful consideration should be given to the histochemical and immunoprecipitation appropriateness of the species and strain procedures; and in immunofluorescence chosen. The investigator should consider and immunoelectron microscopy. the following factors: 1) the quantity of Ab or required (larger animals should Antisera are commonly produced by injec- be considered when larger quantities of Ab tion of the immunogen (antigen) of interest are required); 2) the phylogenetic relation- into an animal, often in combination with an ship between the species from which the adjuvant to increase the immune response. protein antigen originated and the species The antibody response can be enhanced by used to raise the Ab; 3) the effector function subsequent booster injections of the antigen of the pAbs made by the species raising the with or without adjuvant. Blood samples are Ab (such as complement fixing ability – see obtained from the animal to assess the Hanly, et al., 1995); and 4) the intended use level of antibodies produced, and once a of the antibodies (e.g., in an ELISA, the sufficiently high titre has been reached, the antibody which binds to the antigen may antiserum is prepared by blood collection need to be derived from a different species followed by serum preparation, with subse- than the secondary antibody used in the quent purification of antibodies from the next step of the assay). Different strains of a serum if required. species may also react differently, due to genetic variation in presentation of the Background knowledge of the mechanisms antigen in major histocompatibility complex involved in generating a humoral immune molecules and in immune response regula- response is essential in the development of tory mechanisms (Hanly, et al., 1995; the most appropriate SOPs for particular Coligan, et al., 1997).

3 The rabbit is the most commonly used in animal use as chickens produce larger animal for the production of pAbs, as it is quantities of antibodies than laboratory easy to handle and to bleed, and for most rodents (Schade, et al., 1996; Bollen, et al., applications will produce an adequate 1995). Typically, a single egg will contain up volume of high-titre, high affinity, antiserum to 250mg pAb in the egg yolk (Erhard, et al., (Stills, 1994). A typical bleed from a rabbit 2000). In general, chickens are potent should yield approximately 250mg of pAb; a antibody producers and their immunological terminal bleed using saline displacement responsiveness is similar to that of can yield approximately 1g of pAb. It is mammals. However, it is important to important to use disease-free rabbits for emphasize that the chicken is not suitable all immunological procedures to reduce for all applications, and appropriate facilities the likelihood of Pasteurella multocida for housing chickens must be available if ccac guidelines abscesses at injection sites, and to mini- they are to be used. mize the likelihood of cross-reactivity to other antigens the rabbits’ immune systems In general, rodents are used less frequently may previously have encountered. than rabbits for pAb production, but may be suitable when small volumes of pAbs For protocols requiring the production of are required. Blood volumes that can be large volumes of pAbs, or where the collected from these animals are consider- animals are to be maintained as antibody ably smaller, and thus collection of a producers for a long period of time, the reasonable volume may require cardiac use of whiffle balls or serum pockets for puncture under terminal anesthesia. The antibody collection may be considered rat can be used when Abs of restricted (Whary, et al., 1998). Investigators and specificity to mouse proteins are required, ACCs should evaluate whether the subse- or for IgE production (Garvey, et al., 1977). quent ease of handling for serum sampling The hamster is used to produce anti-mouse will outweigh the initial requirement for protein Abs when such pAbs cannot be surgical intervention to implant the whiffle readily produced in the rat or when broader ball chamber. specificity of pAbs is required.

The chicken is phylogenetically distant from Historically, the guinea pig was commonly mammals and, therefore, can be useful for used for antibody production, in particular raising pAbs to mammalian proteins, and for use in insulin assays, but otherwise it in particular, to intracellular mammalian does not appear to have any significant proteins, as the amino acid sequence of advantage over the use of rodents. many intracellular proteins tends to be conserved between mammalian species. Larger species are used when large The product, IgY, is for almost all purposes volumes of antisera are required, in parti- equivalent to the mammalian IgG class of cular for commercial production. Horses, Abs. The chicken may be considered as a sheep and goats, for example, have a long refinement of technique as pAbs can be life span, are relatively easy to handle, extracted from the egg yolk, removing the and can be bled from the jugular vein. necessity for blood collection. The use of Requirement for large animal facilities and the chicken can also represent a reduction the expense of maintaining larger animals

4 has limited their use principally to commer- status of the animals; the training, expertise cial production of antisera. It should be and competence of the animal care staff; noted that horses, which have been used and the diet and housing. In addition, it to produce antisera for toxins and venoms should be noted that the presence of infec- for clinical applications, are particularly tious agents and/or stress can suppress intolerant of oil-based adjuvants, such as the immune response, and thus reduce the

Freund’s Complete Adjuvant (FCA) and quantities of pAbs produced, or even result antibody production, 2002 saponin (Quil A). Antibodies can also be in no significant response. harvested from the milk of cattle, sheep and goats and represent a non-invasive means The pre-immunization status of the animals, to repeatedly acquire large volumes of pAbs with respect to the immunogen, or other (Brussow, et al., 1987; Tacket, et al., 1988; cross-reacting antigens to be administered, and Sarker, et al., 1998). should be determined. Immunogen-specific pre-existing Abs can influence both the Where species-specific considerations are quality and the quantity of the Abs, espe- not an issue, the species should be chosen cially where a monospecific pAb is required. to minimize pain and distress, bearing in Usually a pre-immunization (pre-bleed) mind ease of handling for injection and sample of serum is prepared in sufficient blood sampling. The expected requirement quantity to use as a control throughout of the pAb will influence the volume of testing and subsequent use of the immune serum required and this will impact on the serum. animal species and number of animals used. In general, young adult animals are General guidance on appropriate care for better pAb producers than older animals, as animals to be used in Ab production is immune function peaks at puberty and then provided in other CCAC guidelines. Animals slowly declines. In addition, females are should be housed under conditions that preferred as they often produce a stronger best meet their social and behavioral needs immune response and tend to be more to permit natural behavior, with group docile, easier to handle, and easier to pair housing being preferred (CCAC, 1990). or group house. For larger farm animal Where that is not possible, the decision species, castrated males may also be suit- should be justified to an ACC. able. Grossman (1989) has documented the enhanced production of pAbs by adult Guideline: Investigators, research females. support and animal care staff involved in polyclonal antibody production must Other factors which influence pAb produc- have the appropriate training and tion include: the nature of the immunogen to competence. be used; the route and timing of administra- tion; the type and quality of adjuvant; the The training and competence of the animal nature of the immunogen/adjuvant formula- users and animal care staff are of great tion (i.e., emulsion, liposome formation, importance in minimizing pain and/or immunostimulatory complex [iscom] forma- distress for the animals. In addition to tion or adsorption); the method of blood having knowledge of animal care, the collection; the strain, health and genetic staff should understand basic immuno-

5 logical principles and be trained and be changed to Category of Invasiveness competent in immunization procedures level C following a favorable report on the and in blood collection. For some species, condition of the animals by those respon- this will require knowledge of anesthetic sible for their day to day care. procedures. The staff must also be capable of recognizing pain and/or distress in the species used (CCAC, 1993, 1999). 3. Standard Operating Procedures

Guideline: Each facility should establish 2. Immunization Protocol Standard Operating Procedures for pAb production in each species. Guideline: Animal Care Committees are ccac guidelines expected to evaluate immunization The immunization protocol raises several protocols with respect to animal welfare significant issues that must be considered: aspects; particular attention should be 1) method of antigen preparation; 2) use of given to atypical immunogens and/or adjuvant – whether to use one and then adjuvants likely to cause pain and/or which one; 3) route of injection, and number distress. of sites to be used; 4) volume to be injected; and 5) schedule of injections. SOPs should The proposal for antibody production for also be implemented to limit the frequency each new immunogen or immunogen/ of use of individual animals and to limit the adjuvant combination must be reviewed by length of time that an individual animal the local ACC, and should include reason- can be held for the purpose of antibody able efforts to determine whether the production. product required is available commercially, or from another research group. The review can be brief if the type of immunogen 4. Immunogen Preparation has been used previously and SOPs are already in place. Protocols involving novel Guideline: The immunogen should immunogens should be given a more be prepared in such a manner to elicit careful review, and the advice of an immu- an acceptable response without adverse- nologist should be sought in these ly affecting the well-being of the animals. instances. SOPs should be in place to address the routine procedures, but may The quality of the immunogen prepara- need slight modification depending on the tion must be a prime consideration. The particular proposal. The use of FCA can immunogen must be non-toxic and it must cause considerable pain and distress for be prepared aseptically, or otherwise the animals concerned. Therefore, protocols rendered sterile and free of toxins and which involve the use of FCA, or where pyrogens. In particular, any chemical the investigator anticipates that the animals residues, contaminating endotoxins or other may experience an adverse reaction, must toxic contaminants must be minimized (e.g., receive particular attention and must <1ng/ml for immunogens derived from be assigned to Category of Invasiveness gram -ve ) and the pH must be level D (CCAC, 1991). This category may adjusted within physiological limits. Most

6 protein immunogens can be filter sterilized the immunogen prepared in a water-in-oil through a microporous cellulose acetate emulsion requiring vigorous shaking (Allison filter (0.22µm pore size). In a recent survey & Byars, 1991). Antigen adsorbed to alum of Canadian institutions that routinely can also give a good Ab response to native prepare pAbs (Craig & Bayans, 1999), configuration (Chen, et al., 1964; Dardiri & respondents stated that immunization with Delay, 1964; Butler, et al., 1969; Cohen, et

proteins in polyacrylamide gel was asso- al., 1970; Weeke, et al., 1975; and Ziola, B., antibody production, 2002 ciated with adverse reactions at the site of pers. comm.). injection.

The quantity of immunogen to be injected to 5. Choice of Adjuvant elicit a good antibody response varies greatly depending on the species and strain Guideline: The decision as to whether an of the animal to be immunized, the adjuvant adjuvant is required should be carefully used, the route and frequency of injection considered and justified. Investigators and the immunogenicity of the immunogen should seek the most appropriate adju- itself. vant for the antigen of interest, bearing in mind the current state of knowledge The quality and quantity of the pAb pro- on immunogen/adjuvant preparation. duced is dependent on the size and the The advice of a knowledgeable immunol- state of the immunogen. Small polypep- ogist should be obtained for each tides and non-protein molecules may protocol involving a new adjuvant or require conjugation with a larger immuno- novel immunogen. The use of Freund’s genic carrier protein in order to provoke an Complete Adjuvant should be avoided immune response. Appropriate carrier mole- where possible, and when necessary cules must be selected, so as not to induce should only be used for the primary suppression of the immune response to the immunization, and should contain less antigen (carrier-induced epitopic suppres- than 0.5mg/ml mycobacteria. sion) due to pre-existing antibodies to the carrier (Schutz, et al., 1985). Investigators and ACC members should not overlook the considerable pain and/or It should also be noted that the conforma- distress that adjuvants can cause for tion of the immunogen may be important, as laboratory animals. Most of the undesirable antibodies raised against native proteins side-effects of pAb production that range in may react best with native proteins, and severity and duration are caused by the those raised against denatured proteins adjuvant (Jennings, 1995). Therefore, it may react best with denatured proteins. For should be determined first whether an example, if the pAbs are to be used to block adjuvant is required. Adjuvants are used to an active site on a protein, the immunogen enhance the immune response and when should not be denatured prior to injection. In used should result in enhanced and this regard it should be noted that a sustained Ab levels. preformed oil-in-water emulsion adjuvant with adsorbed immunogen will maintain a Adjuvants work in several ways. They may greater amount of native conformation than form a depot of immunogen at the injection

7 site leading to a slow release over a period calcium phosphate. However, all desirable of time giving sustained stimulation to the characteristics of an adjuvant are not found immune system. The adjuvant can also help together in any of the 100 or so known deliver the immunogen to the spleen and/or adjuvants. In addition to the brief descrip- lymph nodes where many of the cell-cell tions of the commonly used alternatives interactions in the immune response occur. to FCA/FIA used in Canada which are They may directly or indirectly activate provided in Appendix A, see Leenars, et al. the various cells, such as or (1999); Jennings (1995); CEDARLANE T-helper lymphocytes, involved in the Laboratories Limited "The Adjuvant Guide" immune response. Adjuvants may also (Hornby, Ontario), or the European Adju- influence the duration, subclass and avidity vant Database (Stewart-Tull, 1995) for more of the antibodies produced and may affect detailed information. ccac guidelines cell-mediated (Hunter, et al., 1995). The CCAC recognizes that many investi- gators have been reluctant to discontinue The use of an adjuvant is usually necessary the use of FCA because it is used as a for soluble, relatively pure or pure immuno- "gold standard" due to its well known effec- gen preparations. If the immunogen is a tiveness with a wide variety of antigens small molecule, it usually does not (Craig & Bayans, 1999). While FCA is one induce an immune response by itself. of the most effective adjuvants, it can cause The use of an adjuvant can also mean a greater chronic inflammatory response, that less immunogen is required, which and therefore, should be used only when may be a consideration for molecules which there is evidence that other adjuvants are scarce or are particularly valuable. will not work (for example, when only Highly aggregated antigens may not small amounts of soluble immunogens are require the use of an adjuvant and available, or when the antibody response may in fact induce a more authentic is weak, e.g., Jennings, 1995). Broderson immune response in the absence of (1989) has shown that if the concentration adjuvant. of mycobacteria in the FCA preparation is less than 0.1mg/ml, less severe inflam- The use of adjuvant with whiffle balls is matory reactions result. If large amounts not necessary as the whiffle ball itself of a particulate immunogen are available, serves as an immunogen depot, leading to or if the antigen is highly immunogenic, prolonged release of the antigen (Clemons, immunization without adjuvant or with et al., 1992). other adjuvants should be used. If FCA must be used, it should be used only for The immunogen/adjuvant mixture should be the initial subcutaneous immunization, easily injectable in small volumes and following the procedures detailed in Section should have low toxicity. The commonly C.7 – Volume and Number of Injection used adjuvants include both Freund’s Sites. FIA should be used for subsequent Complete and Incomplete Adjuvants (FCA booster immunizations, if an adjuvant is and FIA), Quil A, RibiTM and TiterMaxTM, and required. Secondary (booster) immuniza- mineral-based adjuvants – aluminium tions can sometimes be carried out using a hydroxide, aluminium phosphate and saline buffer solution.

8 In general, an immunogen should be mixed accurately acquire the desired cells. with an equal volume of adjuvant and emul- However, this can be achieved with much sified. Emulsification can best be achieved less distress to the animal by injecting at the using two Luer-Lok syringes and a locking base of the tail, or in the popliteal area. connector, passing the adjuvant and immunogen back and forth until it becomes Intravenous administration can be the route

paste-like. Sonication using a narrow probe of choice for small particulate immunogens antibody production, 2002 is an alternative method of emulsification; because the immunogen is distributed however, care must be taken to avoid over- throughout the body and hence, capture by heating by keeping the sonication mixture lymphoid cells is high. The IV route should on ice. Proper preparation of the immuno- not be used for oil-based or viscous gel gen/adjuvant mixture is important as a adjuvants or for large particulate immuno- major cause of immunization failure is due gens due to the risk of pulmonary embolism to inappropriate emulsification. Dispersal of (Herbert, 1978). the emulsion with an equal volume of 2% Tween 80 reduces the viscosity of the Subcutaneous injections should be used for solution for easier injection (Herbert, 1965). oil or viscous gel adjuvants, in particular for FCA, in order to minimize the formation of sterile abscesses. The IM route should 6. Route of Injection not be used for the injection of oil-based or viscous gel adjuvants in small animals Guideline: The route of injection must be such as mice or rats. Immunogen/adjuvant selected with the objective of causing injected IM can spread along interfascial the least possible distress for the planes between muscle bundles and irritate animal. the nerve bundles leading to serious pathol- ogy due to the inflammatory process. The route of injection may be subcutaneous (SC), intramuscular (IM), intradermal (ID), The ID route should not be used in small intraperitoneal (IP), or intravenous (IV), as animals, and should be restricted to cases detailed in Appendix B. Footpad, intra- where it is absolutely necessary. For rabbits lymph node or intrasplenic routes are or larger animals where the ID route is strongly discouraged and if required used, the volumes administered should be must be justified on a case by case small (i.e., 25µl in rabbits) (Halliday, et al., basis. Injection into any closed space 2000). is painful, so even IM and ID can be questioned as a route of choice. No Intraperitoneal injection of adjuvant mixtures other routes are permitted. Footpad is not recommended for pAb production, injections have been used in the past to since it is known to induce , follow the immune response to a specific peritonitis, and behavioral changes antigen by studying the cells of the immune (Leenars, et al., 1999; Toth, et al., 1989). system responsible for processing the See Section C.7 – Volume and Number of antigen. Immunogens injected into the Injection Sites, and Appendix B for further footpad are processed by the popliteal recommendations, should the IP route be node, making it possible to routinely and required.

9 Sites of injection should not interfere with laboratory chickens, the SC route should be subsequent handling of the animals for used with an injection volume <1ml; at least blood sampling, etc. two immunizations are generally given. A primary vaccination and a booster should If large volumes of pAb are required, the be given before the laying period with an use of whiffle balls should be considered. interval of six weeks for emulsion-type Whiffle balls used in rabbits are perforated adjuvants and four weeks for lipopeptide plastic golf balls, available from a variety adjuvants. The stress induced by handling of commercial suppliers. The balls should can have a negative effect on egg produc- be sterilized with ethylene oxide prior tion; as can the nature of the antigen to implantation. Whiffle balls must be or antibody used. Yolk Ab titres should be implanted using aseptic surgical techniques checked 14 days after the last immuniza- ccac guidelines described by Hillam, et al. (1974) and Reid, tion, if Ab titres begin to decrease, boosters et al. (1992). Strict asepsis must be main- can be given during the laying period at tained during surgery to avoid secondary 4-8 week intervals. Antibody contained in infection of the implant. A minimum of four eggs can be collected throughout the laying weeks post-surgery is required for healing period (about one year). prior to experimental use, during which time animals should be housed individually to Other routes of administration of adjuvant avoid injury. Once prepared, the whiffle include aerosol, oral and intranasal admini- ball can be used both for primary and stration. These routes have advantages for booster injections as well as for serum stimulation of an IgA response, and also collection. may be less stressful for the animals. For examples involving mice, see Shen, et al. Immunization of chickens for pAb produc- (2000); Shen, et al. (2001); McCluskie and tion is comparable to that of rabbits with Davis (2001); McCluskie, et al. (2000); respect to route of injection, the amount of Coste, et al. (2000); Falero-Diaz, et al. antigen used and the kinetics of specific (2000); and Holan, et al. (2000). Examples antibody generation (Haak-Frendscho, involving rats are found in Papp, et al. 1994; Song, et al., 1985). Recommenda- (1999); and Baxi, et al. (2000). For horses, tions for chicken immunization (Schade, et see Nally, et al. (2001); for pigs, see al., 1996), include the use of FCA, Specol, Katinger, et al. (1999); and Tuboly & Nagy (2001); and for poultry, see Sharma (1999). or preferably lipopeptide PCSL (Pam3-Cys- ser-[Lys]4; 250µg). Erhard, et al. (1997) found few side effects from the use of PCSL 7. Volume and Number of in comparison with multiple granulomas after FCA or FCA/FIA treatment. Immuno- Injection Sites gen should be administered in the 10ng- 1mg range (preferably 10-100µg). Injection Guideline: The injection volume should should be IM for young laboratory chickens be kept as small as possible and should (neck or breast muscle), and intramuscular be kept within the recommended limits. injection in the leg should be avoided since it can lead to lameness. Chickens should Recommendations are given in the litera- be at least seven weeks of age. For older ture to limit the number of injection sites.

10 However, in terms of minimizing adverse withdrawn from the subcutaneous space, reactions, it appears to be less stressful to the plunger should be pulled back a few mm the animals in the post-immunization phase, and the needle quickly removed from the to minimize the injection volume and to subcutaneous space. In this manner, use multiple sites. Recommended injec- spillage into the dermal tissue will be tion volumes are given in Appendix B, minimized.

both for immunogen plus adjuvant or antibody production, 2002 immunogen alone. It should be noted that Where there is no indication of reaction to in order to minimize immunization reactions, the initial injection, booster injections given the volumes recommended per site for in the vicinity of the initial site take advan- injection of immunogen with adjuvant are tage of the memory cells established in the smaller than the generally recommended draining lymph nodes. This may minimize volumes for injection. It is necessary to the number of injections needed per animal use large gauge needles (20-21 gauge and/or the number of animals required for for the rabbit), as immunogen/adjuvant an effective immune response. Injection mixtures are quite viscous. Use of a double sites should be sufficiently separated to emulsification as described by Herbert prohibit the coalescing of the inflammatory (1965), reduces the viscosity of the lesions, which may result in disruption of solution enabling a smaller size needle blood supply to the area, with subsequent (26-27 gauge) to be used. formation of draining abscesses, or occa- sionally tissue sloughing. However, if there Some species may require sedation prior are indications of local reactions, booster to injection. This may be particularly injections should be distant from previous important if multiple injection sites are to be injection sites and must never be given at used. the site of a granuloma or swelling induced by previous injections. In all cases, the injection site should be aseptically prepared1 and allowed Animals must be closely monitored immedi- to dry immediately prior to injection of ately following injection for any anaphy- the immunogen or immunogen/adjuvant lactic reactions, both after the primary mixture. injection and after the subsequent booster injections (see Section C.9 – Monitoring To minimize the chance of abscess forma- of Animals). Animals experiencing unreliev- tion when using FCA, it is important to able pain and/or distress must be ensure that all material is injected asepti- euthanized. cally into the subcutaneous space, not ID or IM. In particular, care should be taken not to For whiffle balls, injection of immunogen contaminate the needle track. Once the should be directly into the whiffle ball solution has been injected, and before it is chamber (Clemons, et al., 1992).

The boosting protocol can have a significant 1 Aseptic skin preparation means clipping the fur effect on the result of the immunization. and using a three-stage surgical preparation: surgical soap, alcohol rinse and surgical The time between two immunization steps preparation solution. can influence both the induction of B

11 memory cells and the class switch of B cells 8. Blood Collection (from IgM to other antibody classes and subclasses). In general, a booster can be considered after the antibody titre has Guideline: The selection of blood peaked or begun to decline, and when collection procedure should aim to memory cells can be expected to be minimize stress for the animal and increasing. If the first immunization is should follow current CCAC guidelines. performed without a depot-forming adju- vant, the antibody titre will usually peak The SOP for each pAb preparation should 2-3 weeks after immunization. When a include a section on blood collection. In depot-forming adjuvant is used, a booster this respect, the training and expertise of

ccac guidelines injection can follow at least four weeks the staff carrying out the procedure is after the first immunization. An adjuvant a significant factor (CCAC, 1999). The is not always necessary for booster animals should be kept warm and away immunizations. from environmentally-induced stresses such as noise. The use of organic solvents In most cases, the endpoint of Ab produc- to induce vasodilation is not recommended. tion should be considered as the point when The use of general anesthesia is not the Ab titre has reached an acceptable level necessary for blood collection in rabbits (generally a maximum of two boosters). A and larger animals. It is also not necessary long immunization schedule with repeated for blood collection in rodents using boosting is not generally useful as it may the tail vein or saphenous vein. However, result not only in the production of Abs with terminal anesthesia is required for cardiac increased affinity for the immunogen of puncture, which may be required to obtain interest, but also in production of more Abs sufficient volume. It may be useful to use specific for contaminants in the immunogen local anesthetics, for example EMLATM preparation (if the immunogen is not a cream on rabbit ears, and/or parenteral purified protein or peptide) (Leenars, et analgesics. The needle used should be al., 1999). Multiple boosters should not matched to blood vessel size. Rabbits require the use of adjuvant. In all cases the should be bled by the ear vein using a welfare of the animals must be taken into needle rather than a scalpel blade as account. cutting may cause damage to the blood vessels and surrounding tissues, which may Animals can be rested for long intervals ultimately result in regional necrosis and between boosting. In addition, regular sloughing of ear tissue. The maximum intervals of blood collection, once a blood volume removed should not exceed sufficient serum titre has been reached, 10% of the total blood volume of the animal could facilitate the collection of adequate (approximately 1% body weight), if collected amounts of Abs for immunogens in limited every two weeks, and not more than 15% supply. However, animals must not be of the total blood volume if collected every kept in an Ab production program unneces- four weeks (Morton, et al., 1993; McGuill & sarily. Any animals kept long-term in an Rowan, 1989; Diehl, et al., 2001). animal facility should be under long-term surveillance by a veterinarian. Serum collection from implanted whiffle

12 balls is relatively easy and does not require at the injection sites in particular and use of a local anesthetic. The skin over the for overall health or distress in general. whiffle ball chamber must be aseptically Food and water intake, activity and prepared. A sterile hypodermic needle with general appearance should be monitored. a cotton plug is inserted into the dorsal half of the chamber through one of the holes The staff taking care of the animals should

and the fluid withdrawn through a second have in their SOP a daily checklist for antibody production, 2002 needle connected to a syringe (Whary, et monitoring animals producing pAb with al., 1998). definitive endpoints for stopping the procedure (euthanasia) if the animal For pAb production in chickens, eggs develops unrelievable distress due to the are collected daily (usually one egg is inflammatory response. SOPs should laid per day) and marked for identification. include a requirement for investigators to They can be stored for up to one year at contact the veterinary staff (and for the 4°C prior to antibody (IgY) purification veterinary staff to contact the principal (Haak-Frendscho, 1994; Svendsen, et al., investigator) if injection site lesions, or 1995). evidence of pain and/or distress, are iden- tified in any animals. The veterinary staff Exsanguination must be performed under must be responsible for the implementation non-recovery general anesthesia, using of timely and appropriate assessment of the a technique that results in a maximum animals and for institution of when collection of blood. After exsanguination is required. For immunization induced lesions, complete, the death of the animal must be supportive therapy may include topical assured following current CCAC guidelines cleansing, administration, and/or for euthanasia. analgesic administration. Fluid replacement or nutritional supplements may be required if animals have sustained anorexia or 9. Monitoring of Animals decreased fluid intake. Animals must also be carefully monitored following blood Guideline: Animals must be monitored collection, in particular if the procedure daily, and the Standard Operating is carried out under anesthetic (CCAC, Procedures should include a checklist 1998). for endpoints.

10. Disposition of the Animals The SOP should include a requirement for record keeping. The immunization record should include the agent, route, site or sites, Disposition of the animals must be carried volume, date of injection and body weight of out in accordance with CCAC guidelines the animal. and local ACC regulations. It is the investi- gators’ responsibility to inform the animal Animals may have inflammatory responses care staff when the collection of antibody to the injections of immunogen or immu- has been completed so that animals can nogen/adjuvant mixtures and, therefore, be disposed of as previously agreed in must be monitored daily for responses the protocol. This can include euthanasia,

13 or re-homing where relevant adoption for example phage display techniques and programs are in place. It should be noted expression as recombinant proteins, do not that if FCA is used in meat producing involve the use of animals and are therefore animals, they must not be resold for human not discussed here. consumption. The Canadian Food Inspec- tion Agency2 should be contacted for advice Despite the original comments on the on disposition of animals where other general methodology (in vitro) of hybridoma adjuvants have been used. technology by Köhler and Milstein – "the manufacture of predefined specific anti- bodies by means of permanent tissue cul- ture cell lines is of general interest" and D. MONOCLONAL ANTIBODY "such cells can be grown in vitro in massive ccac guidelines PRODUCTION cultures" – the propagation of mAbs has, for almost all purposes, been carried out in vivo. It was apparent that mAbs could be Monoclonal antibodies (mAb) are antibodies produced by injecting the hybridoma cells which have a single, selected specificity and into the abdominal cavities of different which, usually, are secreted continuously by species of rodents. The subsequent propa- "immortalized" hybridoma cells. A hybri- gation of the hybridoma cells in ascitic fluid doma is a biologically constructed hybrid of offered an easy, economical route to the an antibody-producing lymphoid cell and a production of mAbs. malignant (immortal) myeloma cell. The development of by An alternative method of mAb production, Köhler & Milstein in 1975, provided a means although not routinely used, is the produc- for obtaining large quantities of highly tion of recombinant antibodies in the specific antibodies which has had a milk of genetically modified animals. As the profound impact on disease diagnosis, need for larger quantities of antibodies therapy, and on biomedical research in increases, this approach may be increasingly general. adopted as it would appear that the expres- sion of recombinant mAbs in the milk of There are essentially two major stages in transgenic mice and goats may be much the production of mAbs: 1) the induction greater than in cell culture systems (Young, of antibody producing lymphoid cells in et al., 1998). The creation and use of vivo (immunization) and the selection animals as bioreactors for mAb production of antibody-producing hybridoma cells must be carried out in accordance with the in vitro; and 2) the propagation of selected CCAC guidelines on: transgenic animals hybridoma clones, either in vitro or in vivo. (1997b). Appendix C contains a graphical represen- tation of the stages of mAb production. General guideline: Every attempt should Newer methods for production of mAbs, be made to obtain material already available or to use an in vitro method for production of mAbs. Therefore, any 2 Canadian Food Inspection Agency, National Manager, Veterinary Biologics Section, Ottawa proposed production of monoclonal ON (tel.: 613-225-2342). antibodies using the ascites method

14 requires justification by the investigator to "reduce to an absolute minimum the to the animal care committee. amount of distress imposed on those animals that are still used"). Commercially produced mAbs should be obtained from sources that comply The potential for severe pain and/or distress with CCAC guidelines, or if international in the animals relates to: intraperitoneal (IP) are accredited by AAALAC. For lists of injection of primer (e.g., Pristane or FIA); antibody production, 2002 suppliers of in vitro produced mAbs, see the effect of primer after administration; the http://www.frame.org.uk/Monoclonal_ IP inoculation of hybridoma cells; and the Suppliers.htm. growth of the tumor cells in the abdominal cavity (primary effects) as solid peritoneal The production of mAbs in mice by the plaques that infiltrate the abdominal wall ascites method raises several issues of and/or abdominal organs. Specifically, IP concern regarding the potential for severe injection is believed to cause moderate and unnecessary pain and suffering for the distress; ascites fluid production and tumor animals (Anon., 1989). In recognition of this growth have been reported to cause suffer- fact, a number of in vitro replacements for ing in human patients (Kuhlmann, et al., the rodent ascites method of mAb produc- 1989); and, in addition, the complex patho- tion have been developed. A number of physiological and pathological changes may countries have instituted a ban on routine in cause severe distress for the animal vivo production of mAbs (Shavlev, 1998). A involved (Hendriksen, 1998). While ascites recent report prepared by a US National fluid tapping is believed to relieve some of Research Council Committee, in response the pain and/or distress associated with to a request from the National Institutes of abdominal distension, the immobilization Health, recommended that in vitro mAb and administration of anesthetic prior to production methods should be adopted as ascitic fluid harvesting is an additional routine whenever practical, and that if the source of distress. mouse ascites method is used, it must be justified to an institutional animal care and The CCAC recognizes that it has a respon- use committee (Peterson, 1999). It is now sibility to support ACCs in requiring that generally accepted that the in vitro tech- investigators provide evidence that the use niques of mAb production have progressed of replacement methods are not appropriate to the point where these techniques can be (CCAC guidelines on: animal use protocol used for more than 90% of mAb production review, 1997a). Therefore, the CCAC will (ILAR, 1999). develop and maintain a repository of information on suitable in vitro techniques In this context, it should be noted that for the production of mAbs (Appendix D). It the CCAC Ethics of Animal Investigation is also recognized that, in countries where a (1989) requires investigators to follow the ban on the ascites method has been "Three Rs" of Russell & Burch (1959): implemented, the use of in vitro method- Replacement (of animals with other, non- ology has been stimulated by the creation of sentient material or with animals of lower Centres of Excellence, centralizing the sentience); Reduction (of numbers of ani- production of mAbs in vitro, and providing mals used); and Refinement (of technique, training, expertise and resources for the in

15 vitro techniques. A number of organizations little or no mAb in vivo, where in vitro also offer to produce material by in vitro methods cannot be used, investigators means on a contract basis. should be asked to test new hybridomas in a small pilot study involving 2-3 animals. Ideally, a replacement in vitro system should be equally or less expensive, not General guideline: Clearly defined end- require special culture conditions, produce points and close monitoring of the high concentrations of mAbs, be free from condition of the animals are required to contamination, be re-usable, and require a minimize the potential for pain and/or reasonably short period of time to produce distress. relatively pure mAbs in adequate quantities (Falkenberg, 1993; 1995). Currently, there ccac guidelines In accordance with CCAC guidelines on: are no in vitro systems that meet all of choosing an appropriate endpoint in these criteria, but depending on the experiments using animals for research, quality and quantity of mAbs required, teaching and testing (1998), clear endpoints it is possible for investigators to select an must be developed which minimize pain appropriate in vitro system (for references and/or distress for the animal. A clear to various mAb production methods schedule for monitoring and reporting see Appendix D). If the required mAb must be established, and SOPs must be is not commercially available from a established for each new immunogen/ reputable source, investigators should make adjuvant combination. all reasonable efforts to produce the required mAbs using an in vitro approach. In line with recommendations made 1. Animal Selection and Care in the US and elsewhere, prior to approval of a protocol for ascites production, Guideline: When selecting species and the investigator should show that he or strain of animals for mAb production, she has made a substantial effort to the overall consideration must be to adapt the hybridoma to a culture system minimize pain and/or distress. suitable for scale of mAb production required (e.g., tissue culture flasks, The mice (or rats) used should be the same simple membrane systems, hollow fibre, strain (syngenic) both for immunization to fermentation systems) (Saxby, 1999; produce the hybridoma clone and for subse- Smith & De Tolla, 1999). The production quent mAb production, so that the tissue of mAbs by growth of hybridomas as used is histocompatible. BALB/c mice are ascites tumors should be avoided unless often the animal of choice, as many of the absolutely necessary. parental myeloma cells used in the fusion process are derived from BALB/c mice. The following guidelines have been Retired breeders may offer some advan- elaborated to safeguard the well-being of tages for ascites production due to the animals (principally mice) that continue to previously stretched abdominal musculature be used in mAb production. (Falkenburg, 1998).

As up to 15% of hybridomas may produce SCID mice, although expensive, have been

16 reported to produce fewer non-specific antigen is in limited supply and previous murine antibodies with equivalent yields of attempts at in vivo immunization have specific mAb, thus minimizing interference yielded negative results due to similarity to in immunoassays, etc., (Pistillo, et al., self antigens or to the weak immunogenicity 1992). It should be noted that SCID mice of antigen (Bunse & Henz, 1994). Other require barrier facilities for housing and advantages of this approach are: fewer

extensive (and expensive) procedures for animals are required; the immunization antibody production, 2002 appropriate care. period is 4-5 days, compared to several weeks for traditional in vivo immunization ACCs should ensure that the appropriate (Grimaldi & French, 1995); it is possible to numbers of animals are used. This can be monitor and control the immune response estimated by presuming that mice produce without the influence of the immune regula- approximately 0.5-10mg/ml of mAb in tory mechanisms that occur in vivo; and ascites fluid, and that on average it is mAbs can be generated against agents that possible to harvest 2ml ascites fluid per are toxic to animals. tap (Smith & De Tolla, 1999). These are generalized estimates, and each hybridoma For further details concerning production of must be considered as a unique cell line. hybridoma clones, see Grimaldi & French (1995). For a single-step selection and cloning of hybridomas, see Stebeck, et al. 2. Production of Hybridoma (1996); and Chase, et al. (2001). Clones It is important that prior to immunization, the SOPs for immunization should be devel- methodology for detecting the specific oped based on guidelines for choice of antibody of interest in the mouse sera adjuvant, route of injection and volume and and tissue culture supernatant has been number of injection sites (see Sections C.5- developed, otherwise significant time and C.7). Immunization procedures that do not resources may be wasted later in the mAb require adjuvant and that can be performed development phase. In addition, the use for in short time periods have been shown to the mAb should be carefully considered, so be effective for hybridoma derivation that mAbs that are effective in the final func- (Chase, et al., 2001). The procedures, tional assay can be selected by appropriate which appear to cause less stress to the screening. mice, require multiple boosts in a short time frame, eliminating the requirement for adjuvant. However, rapid immunization may 3. Ascites Production not always yield mAbs of high affinity, a factor which should be considered in choice 3.1 Priming of immunization regimen. Guideline: Freund’s Incomplete Adjuvant As an alternative to in vivo immunization, in (FIA) or other less invasive adjuvant vitro immunization with antigen has been should be used as the intraperitoneal successfully used (Borrebaeck, 1989). This priming agent, with a maximum volume procedure is particularly useful when an of 0.3ml (FIA) administered in one injec-

17 tion. If the use of Pristane is proposed, it 3.2 Contamination should be justified to the institutional animal care committee and the volume Guideline: Hybridomas should be tested should be limited to 0.2ml. for the presence of adventitious viral and mycoplasma agents prior to use As originally described, the ascites method for in vivo propagation of monoclonal of mAb production included the use of antibodies. Pristane (0.5ml injected intraperitoneally) as the chemical used to "prime" the peritoneal Viral contamination is commonly found in cavity of the mouse. The effect of the primer murine specimens inoculated with a murine- is two-fold; it suppresses the immune derived hybridoma. This type of contamina-

ccac guidelines system so that the growth of the hybridoma tion may result in infection of the host cells in the abdominal cavity is not (strongly) animal(s) and in spread of the disease impaired, and it causes a chemical irritation, throughout an animal facility. In addition, which leads to peritonitis and the secretion lymphocytic choriomeningitis , which of serous fluid (Kuhlmann, et al., 1989). A can lead to disease in humans, has also hybridoma cell suspension is injected been isolated from murine tumor cell lines 7-10 days later. (Dykewicz, et al., 1992). Mouse hepatitis virus (MHV) can provoke an immuno- Preliminary results indicate that the use of modulatory response, influencing the analgesics prior to priming with Pristane subsequent production of mAbs. It also may lessen the pain and distress experi- spreads rapidly through an animal facility, ence by the mouse (Fletch, A. & Delaney, affecting other work in progress. The typical K., pers. comm.). testing method is the Mouse Antibody Production (MAP) test, which involves Studies comparing Pristane (at several injecting a sample of tissue in question volumes) and other priming agents, have into mice, and, after an incubation period of shown that the use of FIA has several 3-4 weeks, euthanizing the mice to run advantages over Pristane, including: fewer serological tests for a panel of known indications of stress to the animals; adventitious agents (Jackson, et al., 1999). injection of hybridoma cells can be Molecular tests using polymerase chain performed as early as one day after reaction (PCR) and other related techniques priming; and fewer animals are required will improve testing capabilities and turn- (Gillette, 1987; Mueller, et al., 1986; and around times. Jones, et al., 1990). Trypan blue has also been used as a priming agent (Wu & Kearney, 1980). It should be noted that, 3.3 Hybridoma implantation prior to use, Trypan blue must be dialyzed 6 in fresh glass-distilled water for 48 hours, Guideline: Up to 3 x 10 hybridoma cells replacing the water twice per day, to in a maximum volume of 1.0ml may be reduce low molecular weight impurities. injected into the peritoneal cavity of a Priming with Trypan blue is carried out primed mouse. 24 hours and again one hour prior to injection of the hybridoma cells. Tumor cell lines prone to forming solid

18 tumors produce less ascitic fluid. They can accumulating fluid in the abdomen). Once be optimized for ascites production by the ascites fluid accumulation has resulted performing serial passages of non-attached in obvious abdominal swelling, usually tumor cells that have been selected from 7-10 days, the condition of the animal must mice with solid tumors by peritoneal lavage. be assessed at least twice every 24 hours Selection of hybridoma cell-lines which do at regularly spaced intervals. It must be

not adhere to the tissue culture flask remembered that the rate of hybridoma cell antibody production, 2002 has been shown to reduce the likelihood propagation in the mouse can be quite of formation of solid tumors. For many variable. 6 hybridomas, 3 x 10 cells have been shown to be the maximum necessary to give good 3.5 Ascites tumor growth development of mAbs and ascitic fluid. In some circumstances (e.g., hybridomas produced in 1970s-1980s using first genera- Guideline: The increase in body weight tion myeloma cells), a higher concentration due to the accumulation of ascitic fluid of cells may be needed to produce any in the abdomen and/or tumor growth ascitic fluid. Some rapid proliferating, should not produce pain and/or distress aggressive cell lines may require a lower to the animal; in no case should the concentration for inoculation as a rapidly increase in body weight exceed 20% of developing tumor can lead to severe the normal body weight of age and sex distress and death. matched animals of the same strain.

In accordance with the CCAC guidelines on: 3.4 Monitoring the animals and choosing an appropriate endpoint in experi- endpoints ments using animals for research, teaching and testing (1998), a chart for monitoring Following injection of hybridoma cells, the animals is required with clearly defined routine care should include daily observa- endpoints and reporting lines. Once the tions by appropriately trained staff for the endpoint has been reached, abdominal first week (approximately) and before pressure must be relieved by harvesting of ascites fluid accumulation is evident (as the ascitic fluid – or by humane killing indicated by the swelling of the abdomen). followed by recovery of the ascitic fluid. Any observations of unusual behavior or Mice should be weighed daily, beginning symptoms during this time should be four days after inoculation, to monitor addressed promptly. The CCAC guidelines progression of the ascites tumor by weight on: choosing an appropriate endpoint in gain and to monitor the general health of experiments using animals for research, the animals. A baseline weight measure- teaching and testing (1998) should be used ment of the mouse should be taken on to develop monitoring procedures and an the day of inoculation and this value can appropriate endpoint for the animal. then be used to assist in determining Pertinent signs of distress include: decrease when the mouse reaches an increase of in activity; hunched appearance; ruffled hair 20% body weight (Workman, et al., 1998). coat; respiratory distress; and weight Caution should be used when relying on loss (which may be masked by the weight, as mice are likely to lose body mass

19 at the same time as the tumor is growing. of ascitic fluid may be collected at the first Body condition scores can also be used as (survival) tap. The abdomen of the mouse an indicator of well-being (Ullman-Culleré & should be palpated to determine whether an Foltz, 1999) and can assist in deciding intra-abdominal solid tumor is present, when to terminate the procedure. the presence of a tumor being grounds for humane euthanasia of the animal. Administration of replacement fluids (1-2ml 3.6 Ascites fluid collection subcutaneous) should be considered when large volumes of ascitic fluid are harvested Guideline: Depending on the condition and the animals are not terminated of the mouse, a maximum of two taps of (Jackson & Fox, 1995). the ascitic fluid are permitted, with the ccac guidelines second tap being a terminal procedure. The animals should be closely monitored for Training and experience in tapping or the first 60 minutes and regularly for several draining ascitic fluid is essential. hours following the first tap. Any signs of distress should result in euthanasia of the It should be noted that fluid removal carries animal, according to current CCAC guide- the risk of hemorrhage, oedema and death. lines. For more aggressive cell lines, known to cause significant morbidity, the number of ACCs must ensure that the personnel taps should be limited to one terminal responsible for carrying out these proce- procedure under general anesthesia. dures (listed on the animal use protocol) Anesthesia, as recommended by current have obtained the necessary training prior to conducting the procedure. CCAC CCAC guidelines may be used for the first guidelines on: institutional animal user (non-terminal) tap. However, anesthesia training (1999) mandates the training of any leads to a decrease in blood pressure and personnel prior to the performance of respiratory suppression, which may be animal-based procedures. detrimental to the already compromised mouse. Therefore, it is recommended that mice are placed in an oxygen filled induction chamber for 5-10 minutes, E. REFERENCES followed by mild gaseous anesthesia (isoflurane) to give good immobilization 1. General of the animal for the procedure with rapid recovery. To minimize any bacterial conta- ABBAS, A.K. & LICHTMAN, A.H. (2001). mination, the site of paracentesis should be Basic Immunology. 309 pp. Philadelphia PA: aseptically prepared for the first tap. W.B. Saunders.

It is recommended that a large gauge ABBAS, A.K., LICHTMAN, A. & POBER, J. (2000). Cellular and Molecular Immunology, needle (21-22 gauge) be used for paracen- 4th Edn. 553 pp. Philadelphia PA: W.B. tesis. A large gauge needle permits rapid Saunders. collection of the viscous ascitic fluid, reduc- ing the period of restraint/anesthesia ANDERSON, W.L. (1999). Immunology. required; however, too large a needle can 200 pp. Madison CT: Fence Creek Publishing cause tissue damage. A maximum of 4-5ml (Blackwell).

20 COLIGAN, J.E., KRUISBEEK, A.M., glycoprotein E2 induces an immune response MARGULIES, D.H., et al. (eds.) (1997). in cotton rats. 278(1):234-243. Current Protocols in Immunology. Baltimore BOLLEN, L.S., CROWLEY, A., STODULSKI, MD: John Wiley & Sons Inc. G., et al. (1995). Antibody production in GRIMALDI, C.M. & FRENCH, D.L. (1995). rabbits and chickens immunized with human Monoclonal antibodies by somatic cell fusion. IgG. A comparison of titre and avidity develop-

Institute for Laboratory Animal Research ment in rabbit serum, chicken serum and egg antibody production, 2002 Journal 37(3):125-132 (http://www4.nas.edu/ yolk using three different adjuvants. Journal of cls/ijhome.nsf). Immunological Methods 191:113-120.

JANEWAY, C. Jr. & TRAVERS, P. (1999). BRODERSON, J.R. (1989). A retrospective Immunobiology: The Immune System in review of lesions associated with the use of Health and Disease, 4th Edn. 633 pp. New Freund's Adjuvant. Laboratory Animal Science York NY: Garland Publishing Inc. 39(5):400-405.

KUBY, J. (2000). Immunology, 4th Edn. BRUSSOW, H., HILPERT, H., WALTHER, I., 670 pp. New York NY: W.H. Freeman & Co. et al. (1987). Bovine milk immunoglobulins for (http://www.whfreeman.com/immunology/ to infantile rotavirus gas- CH05/kuby05.htm). troenteritis. Journal of Clinical Microbiology 25:982-986. ROITT, I., BROSTOFF, J. & MALE, D. (1998). Immunology, 5th Edn. 423 pp. London UK: BUTLER, N.R., VOYCE, M.A., BURLAND, Mosby. W.L., et al. (1969). Advantages of aluminum hydroxide adsorbed combined diptheria, SHARON, J. (1998). Basic Immunology, 1st , and pertussis vaccines for immuniza- Edn. 303 pp. Baltimore MD: Williams and tion of infants. British Medical Journal March Wilkins. 15:663. SOMPAYRAC, L. (1999). How the Immune CANADIAN COUNCIL ON ANIMAL CARE System Works. 111 pp. Oxford UK: Blackwell (1989). Policy Statement: Ethics of Animal Science. Investigation. Ottawa ON: CCAC (http://www. ccac.ca/english/gui_pol/policies/policy.htm). 2. Polyclonal Antibody Production CANADIAN COUNCIL ON ANIMAL CARE (1990). Policy Statement: Social and ABBAS, A.K. & LICHTMAN, A.H. (2001). Behavioral Requirements of Experimental Basic Immunology. 309 pp. Philadelphia PA: Animals. Ottawa ON: CCAC (http://www.ccac. W.B. Saunders. ca/english/gui_pol/policies/policy.htm). ALLISON, A.C. & BAYARS, N.E. (1991). CANADIAN COUNCIL ON ANIMAL CARE Immunological adjuvants: Desirable properties (1991). Policy Statement: Categories of and side-effects. Molecular Immunology Invasiveness in Animal Experiments. Ottawa 28:279-284. ON: CCAC (http://www.ccac.ca/english/ ANDERSON, W.L. (1999). Immunology. gui_pol/policies/policy.htm). 200 pp. Madison CT: Fence Creek Publishing CANADIAN COUNCIL ON ANIMAL CARE (Blackwell). (1993). Guide to the Care and Use of BAXI, M.K., DEREGT, D., ROBERTSON, J., Experimental Animals, Vol. 1, 2nd Edn. et al. (2000). Recombinant bovine adenovirus 212 pp. Ottawa ON: CCAC (http://www.ccac. type 3 expressing bovine viral diarrhea virus ca/english/gui_pol/guframe.htm).

21 CANADIAN COUNCIL ON ANIMAL CARE stration of substances and removal of blood, (1998). guidelines on: choosing an including routes and volumes. Journal of appropriate endpoint in experiments for Applied Toxicology 21:15-23. animals used in research, teaching and ERHARD, M.H., MAHN, K., SCHMIDT, P., et testing. 30 pp. Ottawa ON: CCAC (http://www. ccac.ca/english/gui_pol/guframe.htm). al. (2000). Evaluation of various immunization procedures in laying hens to induce high CANADIAN COUNCIL ON ANIMAL CARE amounts of specific egg yolk antibodies. (1999). guidelines on: institutional animal user Alternatives to Laboratory Animals 28:63-80. training. 10 pp. Ottawa ON: CCAC (http:// www.ccac.ca/english/gui_pol/guframe.htm). ERHARD, M.H., SCHMIDT, P., HOFMANN, A., et al. (1997). The lipopeptide, Pam3Cys- CHEN, T.H., LARSON, A. & MEYER, K.F. ser-(Lys)4: An alternative adjuvant to Freund's ccac guidelines (1964). Studies on immunization against Adjuvant for the immunization of chicken to plague. Journal of Immunology 93(4):668. produce egg yolk antibodies. Alternatives to Laboratory Animals 25:173-181. CLEMONS, D.J., BESCH-WILLIFORD, C., STEFFEN, E.K., et al. (1992). Evaluation of a FALERO-DIAZ, G., CHALLACOMBE, S., subcutaneously implanted chamber for anti- RAHMAN, D., et al. (2000). Transmission body production in rabbits. Laboratory Animal of IgA and IgG monoclonal antibodies to Science 42(3):307-311. mucosal fluids following intranasal or paren- COHEN, H., NAGEL, J., VAN DER VEER, M., teral delivery. International Archives of et al. (1970). Studies on tetanus . and Immunology 122(2):143-150. Journal of Immunology 104(6):1417. GARVEY, J.S., CREMER, N.E. & COLIGAN, J.E., KRUISBEEK, A.M., SUSSDORF, D.H. (1977). Methods in MARGULIES, D.H., et al. (eds.) (1997). Immunology, 3rd Edn. Reading MA: W.A. Current Protocols in Immunology. Baltimore Benjamin, Inc. MD: John Wiley & Sons Inc. GROSSMAN, C.J. (1989). Possible under- COSTE, A., SIRARD, J.C., JOHANSEN, K., et lying mechanisms of sexual dimorphism in the al. (2000). Nasal immunization of mice with immune response, fact and hypothesis. virus-like particles protects offspring against Journal of Steroid Biochemistry 34:241-251. rotavirus diarrhea. Journal of Virology 74(19): HAAK-FRENDSCHO, M. (1994). Why IgY? 8966-8971. Chicken polyclonal antibody, an appealing CRAIG, S. & BAYANS, T. (1999). Survey of alternative. Promega Notes Magazine 46:11. adjuvant use for the production of poly- HALLIDAY, L.C., ARTWOHL, J.E., HANLY, clonal antibodies. Canadian Association for W.C., et al. (2000). Physiologic and behavioral Laboratory Animal Science Newsletter assessment of rabbits immunized with 33(3):32-35. Freund’s Complete Adjuvant. Contemporary DARDIRI, A.H. & DELAY, P.D. (1964). Topics of Laboratory Animal Science 30(7):8- Production of high potency anti-Taschen 13. disease serum from rabbits and guinea pigs. HANLY, W.C., ARTWOHL, J.E. & BENNETT, Canadian Journal of Comparative Medicine B.T. (1995). Review of polyclonal antibody 28(1):3. production procedures in mammals and DIEHL, K.-H., HULL, R., MORTON, D., et al. poultry. Institute for Laboratory Animal (2001). A good practice guide to the admini- Research Journal 37:93-118.

22 HERBERT, W.J. (1965). Multiple emulsions: Laboratory Animals 27(1):79-102 (http:// A new form of mineral-oil adjuvant. Lancet altweb.jhsph.edu/publications/ECVAM/ October 16:771. ecvam35.htm).

HERBERT, W.J. (1978). Mineral-oil Adjuvants McCLUSKIE, M.J. & DAVIS, H.L. (2001). and the Immunization of Laboratory Animals. Oral, intrarectal and intranasal immuniza- In: Handbook of Experimental Immunology, tions using CpG and non-CpG oligodeoxy

3rd Edn. (ed. D.M. Weir). Pp. A3, 1-3,15. nucleotides as adjuvants. Vaccine 19:413- antibody production, 2002 Oxford UK: Blackwell Scientific Publications. 422.

HILLAM, R.P., TENGERDY, R.P. & BROWN, McCLUSKIE, M.J., WEERATNA, R.D. & G.L. (1974). Local antibody production against DAVIS, H.L. (2000). Intranasal immunization the murine toxin of Yersinia pestis in a of mice with CpG DNA induces strong golf-ball-induced granuloma. Infection and systemic and mucosal responses that are Immunology 10:458-463. influenced by other mucosal adjuvants and antigen distribution. Molecular Medicine 6(10): HOLAN, V., ZAJICOVA, A., KRULOVA, M., et 867-877. al. (2000). Induction of specific transplantation immunity by oral immunization with allogenic McGUILL, M.W. & ROWAN, A.N. (1989). cells. Immunology 101(3):404-411. Biological effects of blood loss: Implications for sampling volumes and techniques. Institute HUNTER, R.L., OLSEN, M.R. & BENNETT, B. for Laboratory Animal Research Journal 31: (1995). Copolymer Adjuvants and TiterMax™. 5-18. In: The Theory and Practical Application of Adjuvants (ed. D.E.S. Stewart-Tull). New York MORTON, D.B., ABBOT, D., BARCLAY, R., et NY: Wiley. al. (1993). Removal of blood from laboratory mammals and birds: First Report of the JENNINGS, V.M. (1995). Review of adjuvants BVA/FRAME/RSPCA/UFAW Joint Working used in antibody production. Institute for Group on Refinement. Laboratory Animals 27: Laboratory Animal Research Journal 37:119- 1-22. 125. NALLY, J.E., ARTIUSHIN, S., SHEORAN, KATINGER, A., LUBITZ, W., SZOSTAK, M.P., A.S., et al. (2001). Induction of mucosal and et al. (1999). Pigs aerogenously immunized systemic antibody specific for SeMF3 of with genetically inactivated (ghosts) or irradi- Streptococcus equi by intranasal vaccination ated Actinobacillus pleuropneumoniae are using a sucrose acetate isobutyrate based protected against a homologous aerosol delivery system. Vaccine 19:492-497. challenge despite differing in pulmonary PAPP, Z., BABIUK, L.A. & BACA- cellular and antibody responses. Journal of ESTRADA, M.E. (1999). The effect of Biotechnology 73(2-3):251-260. pre-existing adenovirus-specific immunity on KUBY, J. (2000). Immunology, 4th Edn. immune responses induced by recombinant 670 pp. New York NY: W.H. Freeman & Co. adenovirus expressing glycoprotein D of (http://www.whfreeman.com/immunology/ bovine herpesvirus type 1. Vaccine 17: CH05/kuby05.htm). 933-943.

LEENARS, M.P.P.A., HENDRIKSEN, C.F.M., REID, J.L., WALKER-SIMMONS, M.K., DE LEEUW, W.A., et al. (1999). The produc- EVERARD, J.D., et al. (1992). Production tion of polyclonal antibodies in laboratory of polyclonal antibodies in rabbits is simpli- animals: The report and recommendations fied using perforated plastic golf balls. of ECVAM Workshop 35. Alternatives to Biotechniques 12:661-666.

23 RUSSELL, W.M.S. & BURCH, R.L. (1959). System Works. 111 pp. Oxford UK: Blackwell The Principles of Humane Experimental Science. Technique. London: Methuen. 238 pp. Universities Federation for Animal Welfare SONG, C.-S., YU, J.H., BAI, D.H., et al. α (UFAW), Potters Bar, Herts, UK: England. (1985). Antibodies to the -subunit of insulin Special edition (1992) (http://altweb.jhsph. receptor from eggs of immunized hens. edu/publications/humane_exp/het-toc.htm). Journal of Immunology 135(5):3354-3359.

SARKER, S.A., CASSWALL, T.H., ALBERT, STEWART-TULL, D.E.S. (ed.) (1995). The M., et al. (1998). Successful treatment of Theory and Practical Application of Adjuvants. rotavirus diarrhea in children with immuno- 392 pp. New York NY: Wiley. globulin from immunized bovine colostrum. STILLS, H.F. (1994). Polyclonal Antibody Pediatric Infectious Diseases Journal 17:1149- Production. In: The Biology of the Laboratory ccac guidelines 1154. Rabbit, 2nd Edn. (eds. P.J. Manning, D.H. SCHADE, R., STAAK, C., HENDRIKSEN, C., Ringler & C.E. Newcomer). Pp. 435-448. San et al. (1996). The production of avian (egg Diego CA: Academic Press. yolk) antibodies-IgY: The report and recom- SVENDSEN, L., CROWLEY, A., OSTER- mendations of ECVAM Workshop 21. GAARD, L.H., et al. (1995). Development and Alternatives to Laboratory Animals 24(6): comparison of purification strategies for 925-934 (http://altweb.jhsph.edu/publications/ chicken antibodies from egg yolks. Laboratory ECVAM/ecvam21.htm). Animal Science 45:89-96. SCHUTZ, M.P., LECLERC, M., JOLIVER, F., et al. (1985). Carrier induced epitopic suppres- TACKET, C.O., LOSONSKY, G., LINK, H. et sion; a major issue for synthetic vaccines. al. (1988). Protection by milk immunoglobulin Journal of Immunology 135:2319-2322. concentrate against oral challenge with enterotoxigenic Escherichia coli. The New SHARMA, J.M. (1999). Introduction to poultry England Journal of Medicine 318(19):1240- vaccines and immunity. Advanced Veterinary 1243. Medicine 41:481-494. TOTH, L.A., DUNLAP, A.W., OLSON, G.A., et SHARON, J. (1998). Basic Immunology, 1st al. (1989). An evaluation of distress following Edn. 303 pp. Baltimore MD: Williams and intraperitoneal immunization with Freund's Wilkins. Adjuvant in mice. Laboratory Animal Science SHEN, X., LAGERGARD, T., YANG, Y., et al. 39(2):122-126. (2000). Preparation and preclinical evaluation TUBOLY, T. & NAGY, E. (2001). Construction of experimental group B Streptococcus type III and characterization of recombinant porcine polysaccharide-cholera toxin B subunit adenovirus 5 expressing the conjugate vaccine for intranasal immunization. transmissible gastroenteritis virus spike Vaccine 19(7-8):850-861. gene. Journal of General Virology 82(Pt.1): SHEN, X., LAGERGARD, T., YANG, Y., et al. 183-190. (2001). Group B Streptococcus capsular poly- WEEKE, B., WEEK, E. & LOWENSTEIN, H. saccharide-cholera toxin B subunit conjugate (1975). The adsorption of serum proteins to vaccines prepared by different methods aluminum hydroxide gel examined by means for intranasal immunization. Infection and of quantitative immunoelectrophoresis. Immunity 69(1):297-306. Scandinavian Journal of Immunology 4 SOMPAYRAC, L. (1999). How the Immune (Suppl. 2):149.

24 WHARY, M.T., CAREY, D.D. & FERGUSON, training. 10 pp. Ottawa ON: CCAC (http:// F.G. (1998). Reduction in animal numbers www.ccac.ca/english/gui_pol/guframe.htm). required for antisera production using the CHASE, J.C., DAWSON-COATES, J.A., subcutaneous chamber technique in rabbits. HADDOW, J.D., et al. (2001). Analysis of Laboratory Animals 32:46-54. kudoa thyrsites (Myxozoa: Myxosporea) spore antigens using monoclonal antibodies. 3. Monoclonal Antibody Diseases of Aquatic Organisms 45:121-129. antibody production, 2002 Production DYKEWICZ, C.A., DATO, V.M., FISHER- HOCH, S.P., et al. (1992). Lymphocytic ANON. (1989). Code of Practice for the choriomeningitis outbreak associated with Production of Monoclonal Antibodies. 6 pp. nude mice in a research institute. Journal of Rijswijk, The Netherlands: Veterinary Health the American Medical Association 267:1349- Inspectorate. 1353.

BORREBAEK, C.A.K. (1989). Strategy for the FALKENBURG, F.W. (1998). Monoclonal production of human monoclonal antibodies antibody production: Problems and solutions. using in vitro activated B cells. Journal of Research Immunology 149:542-547. Immunological Methods 123:157-165. FALKENBERG, F.W., HENGELAGE, T., BUNSE, R. & HEINZ, H.-P. (1994). Charac- KRANE, M., et al. (1993). A simple and terization of a monoclonal antibody to the inexpensive high density dialysis tubing cell capsule of Haemophilus influenzae type b, culture system for the in vitro production of generated by in vitro immunization. Journal of monoclonal antibodies in high concentrations. Immunological Methods 177:89-99. Journal of Immunological Methods 165: 193-206. CANADIAN COUNCIL ON ANIMAL CARE (1989). Policy Statement: Ethics of Animal FALKENBERG, F.W., WEICHERT, H., Investigation. Ottawa ON: CCAC (http://www. KRANE, M., et al. (1995). In vitro production of ccac.ca/english/gui_pol/policies/policy.htm). monoclonal antibodies in high concentrations in a new and easy to handle modular minifer- CANADIAN COUNCIL ON ANIMAL CARE mentor. Journal of Immunological Methods (1997a). guidelines on: animal use protocol 179:13-29. review. 12 pp. Ottawa ON: CCAC (http://www. ccac.ca/english/gui_pol/guframe.htm). GILLETTE, R.W. (1987). Alternatives to pris- tane priming for ascitic fluid and monoclonal CANADIAN COUNCIL ON ANIMAL CARE antibody production. Journal of Immunological (1997b). guidelines on: transgenic animals. Methods 99:21-23. 12 pp. Ottawa ON: CCAC (http://www.ccac.ca/ english/gui_pol/guframe.htm). GRIMALDI, C.M. & FRENCH, D.L. (1995). Monoclonal antibodies by somatic cell fusion. CANADIAN COUNCIL ON ANIMAL CARE Institute for Laboratory Animal Research (1998). guidelines on: choosing an appropriate Journal 37(3):125-132 (http://www4.nas.edu/ endpoint in experiments using animals for cls/ijhome.nsf). research, teaching and testing. 30 pp. Ottawa HENDRIKSEN, C.F.M. (1998). A call for ON: CCAC (http://www.ccac.ca/english/ a European prohibition of monoclonal antibody gui_pol/guframe.htm). production by the ascites procedure in labora- CANADIAN COUNCIL ON ANIMAL CARE tory animals. Alternatives to Laboratory (1999). guidelines on: institutional animal user Animals 26:523-540.

25 INSTITUTE FOR LABORATORY ANIMAL PETERSON, N.C. (1999). In vitro antibodies RESEARCH (ILAR) (1999). Monoclonal and good faith efforts: An overview of the NRC Antibody Production: A Report of the report on monoclonal antibody production. Lab Committee on Methods of Producing Animal Autumn special edition:5-8. Monoclonal Antibodies. ILAR, NRC. PISTILLO, M.P., SYUERSO, V. & FERRARA, Washington DC: National Academy Press G.B. (1992). High yields of anti-HLA human (http://grants.nih.gov/grants/policy/ monoclonal antibodies can be provided by antibodies.pdf). SCID mice. Human Immunology 35:256-259. JACKSON, L.R. & FOX, J.G. (1995). RUSSELL, W.M.S. & BURCH, R.L. (1959). Institutional policies and guidelines on The Principles of Humane Experimental adjuvants and antibody production. Institute Technique. London: Methuen. 238 pp. for Laboratory Animal Research Journal Universities Federation for Animal Welfare ccac guidelines 37(3):141-152 (http://www4.nas.edu/cls/ (UFAW), Potters Bar, Herts, UK: England. ijhome.nsf). Special edition (1992) (http://altweb.jhsph. JACKSON, L.R., TRUDEL, B.A. & LIPMAN, edu/publications/humane_exp/het-toc.htm). N.S. (1999). Small scale monoclonal antibody SAXBY, S.J.Y. (1999). Perspective on in vitro production in vitro: Methods and resources. production of monoclonal antibodies. Lab Lab Animal 28(3):38-50. Animal Autumn special edition:9-11. JOHNSON, D.R. (1995). Murine Mono- SHAVLEV, M. (1998). European and US regu- clonal Antibody Development. In: Methods lation of monoclonal antibodies. Lab Animal in Molecular Biology, Vol. 51: Antibody 27(2):15-17. Engineering Protocols (ed. S. Paul). Pp. 123- 137. Totawa NJ: Humana Press. SMITH, J.M. & DE TOLLA, L.J. (1999). An IACUC guide to reviewing protocols calling for JONES, S.L., COS, J.C. & PERSON, J.E. monoclonal antibody production by mouse (1990). Increased monoclonal antibody ascites. Lab Animal Autumn special edition: ascites production in mice primed with 12-17. Freund’s Incomplete Adjuvant. Journal of Immunological Methods 129:227-231. SPICER, S.S., SPIVEY, M.A., ITO, M., et al. (1994). Some ascites monoclonal antibody KÖHLER, G. & MILSTEIN, C. (1975). preparations contain contaminants that bind to Continuous cultures of fused cells secreting selected Golgi zones and mast cells. Journal antibody of predefined specificity. Nature 256: of Histochemistry & Cytochemistry 42:213- 495-497. 221.

KUHLMANN, I., KURTH, W. & RUHDEL, I. STEBECK, C.E., FREVERT, U., MOMMSEN, (1989). Monoclonal antibodies in vivo and in T.P., et al. (1996). Molecular characterization vitro production on a laboratory scale, with a of glycosomal NAD+-dependent glycerol 3- consideration of the legal aspects of animal phosphate dehydrogenase from Trypanosoma protection. Alternatives to Laboratory Animals brucei rhodesiense. Molecular and Biochem- 17:73-82. ical Parasitology 76:145-158.

MUELLER, U.W., HAWES, C.S. & JONES, ULLMAN-CULLERÉ, M.H. & FOLTZ, C.J. W.R. (1986). Monoclonal antibody production (1999). Body condition scoring: A rapid and by hybridoma growth in Freund’s Adjuvant accurate method for assessing health status in primed mice. Journal of Immunological mice. Laboratory Animal Science 49(3):319- Methods 87:193-196. 323.

26 WORKMAN, P., TWENTYMAN, P., ascites production of monoclonal antibodies. BALKWELL, F., et al. (1998). United Kingdom National Agricultural Library, Beltsville MD. Coordinating Committee on Research Animal Welfare Information Center Newsletter (UKCCCR) guidelines for the welfare of 8(3-4):1-2, 15-18. animals in experimental neoplasia, 2nd Edn. July 1997. British Journal of Cancer 77:1-10. 3.2 Additional useful information

WU, R.L. & KEARNEY, R. (1980). Specific General information on mAbs (http://altweb. antibody production, 2002 tumor immunity induced with mitomycin jhsph.edu/topics/mabs/mabs.htm). C-treated syngeneic tumor cells (MCT). Effects of carrageenan and trypan blue on Lists of suppliers of in vitro produced mAbs MCT-induced immunity in mice. Journal of the (http://www.frame.org.uk/Monoclonal_ National Cancer Institute 64(1):81-87. Suppliers.htm).

YOUNG, M.W., MEADE, H., CURLING, J.M., Nature Biotechnology Guide (http://www. et al. (1998). Production of recombinant guide.nature.com/). antibodies in the milk of transgenic antibodies. University of California, Davis CA - Readings Research Immunology 149:609-610. and Resources for mAbs (http://www.vetmed. ucdavis.edu/Animal_Alternatives/biblio~1. 3.1 Other useful references htm).

APPELMELK, B.J., VERWEIJ-VAN VUGHT, A.M., MAASKANT, J.J., et al. (1992). Murine ascites fluids contain varying amounts of F. GLOSSARY an inhibitor that interferes with comple- ment mediated effector functions of mono- Adjuvant: A substance which increases clonal antibodies. Immunological Letters 33: the immune response to an antigen when 135-138. it is administered at the same time and at the same site as the antigen. When FROMER, M.J. (1997). NIH denies petition to adjuvants are used, smaller doses of the ban in vivo mAb production: Lawsuit threat- antigen are required and the antibody ened. Oncology Times 19:37-40. response persists for a longer period of time. MARX, U. & MERZ, W. (1995). In Vivo and In Vitro Production of Monoclonal Antibodies. Affinity: The strength and stability of Bioreactors versus Immune Ascites. In: the bond between an epitope and antibody Methods in Molecular Biology, Vol. 45, as measured by the amount of antibody- Monoclonal Antibody Protocols (ed. W.C. antigen complex found at equilibrium. Davis). Pp. 169-176. Totowa NJ: Humana Press. Antibody: A protein which is produced in response to a particular antigen and whose MARX, U., EMBLETON, J.M., FISCHER, R., unique structure gives it the capacity to et al. (1997). Monoclonal antibody production: combine specifically with that antigen. The report and recommendations of ECVAM Antibodies are secreted by lymphocytes Workshop 23. Alternatives to Laboratory and are divided into five classes: IgG, IgM, Animals 25(2):121-137 (http://altweb.jhsph. IgA, IgE, and IgD. edu/publications/ECVAM/ecvam23.htm). Monoclonal Antibody: antibody mole- MCARDLE, J. (1997/98). Alternatives to cules which are produced by cells of one

27 clone and recognize only one epitope on is capable of combining with only one an antigen. antibody molecule, but a single antigen may have more than one epitope. Polyclonal Antibody: antibody mole- cules produced by different families of B Granuloma: Chronic inflammatory lymphocytes and consequently recog- response maintained by persistent stimula- nize more than one epitope, even on a tion. single antigen. Histocompatibility: The degree of genetic Antigen: Any substance which can bind to similarity between the donor and receiver of an antibody raised against it. a graft.

Antiserum: The serum, or non-cellular Major Histocompatibility Complex: the

ccac guidelines components of blood which remain after genes responsible for the rejection of clotting, which results from the injection of grafts between individuals. antigens and contains the antibodies produced in response to those antigens. Hybridoma: A biologically constructed hybrid of an antibody-producing lymphoid Ascitic Fluid (also called ascites): An cell and a malignant (immortal) myeloma intraperitoneal fluid extracted from animals cell. that have developed a peritoneal tumor. Immune Response: A humoral or cell- Avidity: The strength of the bond between mediated response of the immune system an antigen and antibody. Avidity depends to an antigen. on the affinity as well as the valences of the antigen and antibody. Humoral Response: the generation of antibodies in response to the presence Clone: Genetically identical cells derived of an antigen. from the same cell. Cell-Mediated Response: the binding of Monoclonal: derived from the same a particular kind of T , known clone and therefore all genetically as a cytotoxic T lymphocytes, with identical. foreign or infected cells, followed by lysis of these cells. Polyclonal: derived from different clones. Immunoblotting: A technique used to identify characteristics of protein antigens. A Complement: A set of plasma proteins that Western blot is a blot of protein onto nitro- act together to attack extracellular forms of cellulose paper followed by detection of pathogens. Complement can be activated specific proteins using labeled antibodies. spontaneously on certain pathogens, or by antibody binding to the pathogen. Immunogen: A substance which can stimulate the immune system. ELISA (Enzyme Linked Immunosorbent Assay): Immunological analysis using Immunogen/Adjuvant Formulation: antigens or antibodies marked by an enzyme. Emulsion: stable oil-in-water or water- in-oil mixture. Epitope: The part of an antigen which is recognized by an antibody. An epitope Liposome Formation: formation of a

28 spherical particulate in an aqueous Memory Cells: Lymphocytes that are able medium by a lipid bilayer enclosing an to quickly and intensively respond to a new aqueous compartment. antigen due to previous exposure to the antigen. Immunostimulatory Complex (iscom) Formation: complex of antigen held Plasma Cells: Specialized cells that are within a lipid matrix that acts as an derived from B lymphocytes and synthesize adjuvant and enables the antigen to be antibodies. antibody production, 2002 taken up into the cytoplasm of a cell after fusion of the lipid within the plasma SCID: Severe Combined Immunodeficient. membrane.

Adsorption: attraction and retention of Based on definitions from: other material on the surface. BACH, J.-F. (1993). Traité d'immunologie. Immunogenicity: The ability to elicit an Collection Médecine-Sciences. 1205 pp. immune response. Paris: Flammarion.

Immunoglobulin: A member of a group of GENETET, N. (1997). Immunologie, 3e éd. proteins having antibody properties, regard- Collection Biologie Médicale. 604 pp. less of whether or not its binding target is Rennes: Ministère de l'éducation nationale, known. (To be considered an antibody, the Éditions Médicales Internationales. antigen with which it binds must be known.) GODING, J.W. (1986). Monoclonal Anti- In mammalian species there are antibodies bodies: Principles and Practice. 2nd Edn. to one of five immunoglobulin (Ig) classes: 315 pp. London UK: Academic Press. IgM; IgG; IgA; IgD; or IgE. In avian species they belong to one of three classes: IgM; HARLOW, E. & LANE, D. (1988). Antibodies: IgY or IgA. A Laboratory Manual. 726 pp. Cold Spring Harbor NY: Cold Spring Harbor Laboratory. , Immunoprecipi- tation, Immunofluorescence, Immuno- REGNAULT, J.-P. (1988). Immunologie electron Microscopy: Techniques for générale. 469 pp. Montréal: Descarie Éditeur. detection or separation of molecules relying on binding of the molecules with specific antibodies, often labeled with fluorescent dyes, etc., in order to visualize the location February 15, 2002 of the molecules.

Lymphocytes: Cells which circulate through the lymph and blood and play a role in immunity. They are subdivided into B lymphocytes and T lymphocytes. For more information on these and B Lymphocytes: produce antibodies other guidelines contact: and their precursors. Canadian Council on Animal Care T Lymphocytes: act as the basis of cell- 315-350 Albert Street mediated immunity and assist B lympho- Ottawa ON CANADA K1R 1B1 cytes in the production of antibodies. (Website: www.ccac.ca)

29 ccac guidelines

30 APPENDIX A terial products (trehalose 6,6'-dimycolate and cell wall skeleton) and a purified gram- COMMON ADJUVANTS negative bacterial product (monophosphoryl lipid A). For a further explanation of TM the biological activities of Ribi adjuvants TM This is a list of the more commonly-used see Jennings (1995). Ribi adjuvants are

adjuvants and their basic properties. It usually less potent than FCA, but they are antibody production, 2002 is not an endorsement of any specific also less toxic and have provided satisfac- product. tory adjuvant activity for many purposes.

Freund’s Complete Adjuvant (FCA) is a Monophosphoryl lipid A (one of the water-in-oil emulsion of mineral oil, mannide TM components of the Ribi system) is by mono-oleate, and heat-killed Mycobac- itself a potential adjuvant option. As with terium tuberculosis (or M. butyricum) or aluminum hydroxide, the safety profile components of the organism. FCA is a of MPL is well documented and provides potent adjuvant that stimulates both excellent adjuvant activity for some humoral and cell-mediated immunity. FCA immunogens. is often strongly reactive as the mineral oil cannot be metabolized and the mycobac- TM TiterMax Adjuvant relies on a micro- terial components can elicit severe granulo- particulate water-in-oil emulsion formed with matous (inflammatory) reactions. The a non-ionic block copolymer (CRL-8941) concentration of mycobacteria varies greatly and squalene, a metabolizable oil. CRL- among commercial preparations of FCA. In 8941 is coated with silica particles which this regard, it should be noted that if the stabilize the emulsion. Stability is a key concentration of mycobacteria is less than TM property of TiterMax as it enables the 0.5mg/ml (0.1mg/ml in Broderson [1989]), emulsion to contain a wide variety of less severe inflammatory reactions result. antigens without the use of large amounts Freund’s Incomplete Adjuvant (FIA) is the of toxic emulsifying agents. The copolymer same as FCA minus the mycobacterial cells enables more antigen to be carried on its or cellular components. FIA is less effective surface than if the antigen were in solution. than FCA for inducing high antibody titres It also activates complement, assisting in and enhancing cell-mediated immunity. retention of the antigen in the lymphoid tissues and activation of immunoreactive TM Ribi Adjuvants are oil-in-water emulsions cells. TiterMaxTM induces increased expres- in which the antigen is blended with a sion of class II (Ia) major histocompatibility minimal volume of oil and then emulsified in complex molecules (MHC) on macro- a saline solution containing the surfactant phages, and increases the presentation of Tween 80 (Ribi, et al., 1975). Although Ag to T cells. When TiterMaxTM works well, it oil-in-water emulsions are less viscous and can induce antibody titers as high or higher easier to inject than water-in-oil emulsions, than FCA and exhibits less toxicity. they are poor adjuvants alone, requiring However, adjuvantation with TiterMaxTM immunostimulants to increase the immuno- is not always successful. In addition, genicity. The immunostimulants used in the TiterMaxTM contains small quantities of an TM Ribi system include two refined mycobac- emulsifying agent and silica particles which

31 have been implicated in delayed-type CpG DNA is a non-specific immune activa- vaccine reactions. tor that can be used alone or in combination with other adjuvant components to augment Quil A is a partially purified form of saponin immune responses. Commercial prepara- or triterpene glycoside derived from the bark tions of CpG DNA comprise short pieces of the Quila saponara tree and purified to of DNA (oligonucleotides) that contain reduce the presence of components which unmethylated cytosine-guanine dinucleo- cause adverse local reactions. In general, tides within a certain base contact. The saponins should not be injected intraperi- mammalian immune system has evolved to toneally or intravenously due to their recognize these sequences, which are hemolytic activity. As Quil A is only found naturally in bacterial DNA, as a sign semi-purified, it can still induce significant of infection. Different CpG DNA sequences ccac guidelines pyrogenic and local reactions. Therefore, activate the immune systems of different prior to using a formulation containing Quil species, and the commercial prepara- A, it should be evaluated in vitro (i.e., for tions of this adjuvant are therefore species- cell culture toxicity). The adjuvant properties specific. of Quil A rely on the formation of iscoms (immune stimulating complexes) which are Additional Information on Adjuvants especially useful in inducing Abs to membrane antigens. The iscoms are 35nm The Adjuvant Guide: CEDARLANE Labora- clathrate bodies like micelles made of Quil tories Limited (http://www.cedarlanelabs. A, cholesterol, phosphatidyl choline and com). antigen. The purified saponin component Animal Welfare Information Center, Infor- which has the highest adjuvant/toxicity ratio mation Resources for Adjuvant and Antibody is called QS21. Saponin containing formula- Production: Comparisons and Alternative tions should never be administered via Technologies (1990-97) (http://www.nal. mucosal surfaces, such as intranasal when usda.gov/awic/pubs/antibody/). induction of IgA is desired. LEENARS, P.P.A.M. (1997). Adjuvants in Laboratory Animals. 207 pp. Rotterdam, The Mineral-Based Adjuvants. Three mineral Netherlands: Erasmus University. compounds are generally used in adjuvants: aluminum hydroxide; aluminum phosphate; RIBI, E.T., MEYER, J., AZUMA R., et al. (1975). Biologically active components and calcium phosphate. Aluminium salts from mycobacterial cell walls IV. Protection are claimed to be superior to all other adju- of Mice Against Aerosol Infection with vants in their ability to increase immune Virulent Mycobacterium tuberculosis. Cellular responses against weak immunogens, Immunology 16:1-10. including those for which FCA does not work. Alhydrogels are sterile aluminium STEWART-TULL, D.E.S. (ed.) (1995). The hydroxide gels that are pyrogen-free and Theory and Practical Application of Adjuvants. stable, they have a high adsorptive capa- 392 pp. New York NY: Wiley. city. At pH <9 alhydrogels have a positive WEERATNA, R.D., McCLUSKIE, M.J., XU, Y. charge so they readily adsorb negatively et al. (2000). CpG DNA induces stronger charged molecules (e.g., proteins at neutral immune responses with less toxicity than pH). other adjuvants. Vaccine 18:1755-1762.

32 APPENDIX B

IMMUNIZATION – RECOMMENDED ROUTES AND VOLUMES (adapted from Leenars, et al., 1999) antibody production, 2002 TABLE I SUGGESTED ROUTES OF INJECTION WITH OR WITH0UT ADJUVANT

Primary Injection Booster Injection(s) with adjuvant without adjuvant with adjuvant without adjuvant

SC IV SC SC IM+ SC IM+ IM ID‡ IM ID‡ IV IP* IP IP ID‡ ID‡

* Only in mice, and not recommended in general. + Not to be used for viscous adjuvants in small animals. ‡ Not to be used in small animals, and not recommended in general.

TABLE II MAXIMUM VOLUMES FOR INJECTION OF IMMUNOGEN/ DEPOT FORMING ADJUVANT MIXTURES PER SITE OF INJECTION FOR DIFFERENT ANIMAL SPECIES

Maximum Volume Primary Subsequent Species Per Site Injection Injections

Mice, hamsters 100µl (0.1ml) SC SC Mice, hamsters 50µl (0.05ml) IM+ IM+ Mice 500µl (0.5ml) IP* SC, IM+ Guinea pigs, rats 200µl (0.2ml) SC, IM+ SC, IM+ Rabbits 250µl (0.25ml) SC, IM SC, IM 25µl (0.025ml) ID SC, IM Sheep, goats, donkeys, pigs 500µl (0.5ml) (if in multiple SC, IM sites, 250µl [0.25ml]/site) Chickens 500µl SC, IM

* Not recommended in general for pAb production. + Not recommended in general, in particular not for viscous adjuvants.

33 TABLE III RECOMMENDED MAXIMUM VOLUME OF INJECTION USED FOR ANTIGEN WITHOUT ADJUVANT FOR DIFFERENT ANIMAL SPECIES

Maximum injection volume in mls

SC IM ID IP IV

Mice 0.5ml 0.05ml 1ml 0.2ml Hamsters 1.0ml 0.1ml 2-3ml 0.3ml Guinea pigs 1.0ml 0.1ml 10ml 0.5ml Rats 1.0ml 0.1ml 5ml 0.5ml ccac guidelines Rabbits 1.5ml 0.5ml 0.05ml 20ml 1-5ml Sheep or goats 5.0ml 0.05ml 10ml 30ml Pigs (<50kg) 3.0ml 2.0ml 0.1ml 250ml 20ml Chickens 4.0ml 0.5ml 10ml 0.5ml

These volumes are suggested maximum 124. Cold Spring Harbour NY: Cold Spring volumes, additional information can be Harbor Laboratory. found in the following texts: HERBERT, W.J. & KRISTENSEN, F. (1986). BAUMANS, V., TENBERG, R.G.M., Laboratory animal techniques for immunology. BERTENS, A.P.M.G., et al. (1993). Experi- In: Handbook of Experimental Immunology, mental Techniques. In: Principles of Labora- Vol. 1, Immunology – Laboratory Manuals (ed. tory Animal Science (eds. L.F.M. van Zutphen, D.M. Weir). Pp. 133.1-133.13. Oxford UK: V. Baumans & A.C. Beynen). Pp. 299-318. Blackwell. Amsterdam, The Netherlands: Elsevier.

DIEHL, K.-H., HULL, R., MORTON, D., et al. LEENARS, M.P.P.A., HENDRIKSEN, C.F.M., (2001). A good practice guide to the admini- DE LEEUW, W.A., et al. (1999). The produc- stration of substances and removal of blood, tion of polyclonal antibodies in laboratory including routes and volumes. Journal of animals: The report and recommendations of Applied Toxicology 21:15-23. ECVAM Workshop 35. Alternatives to Labora- tory Animals 27(1):79-102 (http://altweb. HARLOW, E. & LANE, D. (1988). Adjuvants. jhsph.edu/publications/ECVAM/ecvam35. In: Antibodies: A Laboratory Manual. Pp. 96- htm).

34 APPENDIX C 1. The immunogen is injected into the animals, often, but not always, in combi- nation with an adjuvant to enhance the STAGES OF MONOCLONAL immune response (see Sections C.2- ANTIBODY PRODUCTION C.7 – Immunization Protocol, Standard Operating Procedures, Immunogen

Formation and Selection of the Hybri- Preparation, Choice of Adjuvant, Route antibody production, 2002 doma Clone of Injection, and Volume and Number of Injection Sites). The first stage (formation and selection of hybridoma clones) generally involves the 2. In general, the interval between booster use of one or more mice or rats. When doses of immunogen should be a using mice, it is recommended that they be minimum of 7-10 days, unless a rapid 6-8 weeks of age and free of any concurrent immunization protocol without adjuvant infections. is used.

Production of monoclonal antibodies on a laboratory scale – in vivo (mouse) and in vitro (cell culture) methods

Reproduced with permission from Kuhlmann, I., Kurth, W. & Ruhdel, I. (1989). Monoclonal antibodies: In vivo and in vitro production on a laboratory scale, with a consideration of the legal aspects of animal protection. Alternatives to Laboratory Animals 17:73-82.

35 3. Test bleeds should be performed three bolic defect which prevents them from days after the last booster to ensure using the bypass pathway. Conse- that there is an appropriate response to quently, myeloma cells die in HAT the antigen and that specific antibodies medium. The spleen cells will later die are being produced. Most immunolo- naturally in culture after 1-2 weeks, but gically-based assays for determining the fused cells will continue to survive whether the desired Abs are being pro- as they have the immortality of the duced require less than 10µl of mouse myeloma cells and the metabolic serum. Once an appropriate response bypass of the spleen cells. Some of the has been confirmed, the mouse should fused cells will also have the antibody be boosted again; three days after the producing capacity of the spleen cells. boost, the mouse should be euthanized ccac guidelines and the spleen harvested. Lymphoid 6. Immunoassay procedures are used to cells are then isolated from the spleen, screen for hybridomas secreting the and in some cases from the lymph desired antibody. If positive, the nodes. cultures are cloned. This is accom- plished by plating out the cells so that 4. The lymphoid cells are fused with only one cell is in each well. This parental myeloma cells grown in vitro, produces a clone of cells derived from a by the addition of polyethylene glycol to single progenitor which is both immortal promote membrane fusion. Only a small and produces antibodies. proportion of these cells fuse success- fully. 7. The selected hybridoma cells are often cloned a second time or third time in 5. The mixture of the two unfused cell vitro to ensure cultures of truly mono- types and the newly formed hybrids clonal hybridomas with a single is cultured in a selective cell culture antibody specificity are produced (see medium containing HAT. HAT is a Section D.3.2 – Contamination). At this mixture of hypoxanthine aminopterin stage, cells are grown to larger and thymidine. One of the components numbers in order to prepare for cryop- of HAT, aminopterin, is a powerful toxin reservation. which blocks a metabolic pathway. This pathway can be bypassed if the cell 8. The propagation of cloned hybridoma is provided with the intermediate cells can be accomplished either by metabolites hypoxanthine and thymi- continuing to grow the cells in vitro, or dine. While spleen cells can use this by propagating them in vivo. For bypass pathway and grow in HAT information on in vitro production medium, myeloma cells have a meta- systems see Appendix D.

36 APPENDIX D the production of monoclonal antibodies from hollow fibre bioreactors. 576-587.

DE GEUS, B. The next generation of INFORMATION ON IN VITRO antibodies: Production of recombinant TECHNIQUES FOR antibodies or fragments derived thereof. MONOCLONAL ANTIBODY 587-589. antibody production, 2002 PRODUCTION FRENKEN, L.G.J., HESSING, J.G.M., VAN DEN HONDEL, C.A.M.J.J., et al. Recent advances in the large-scale 74th Forum in Immunology. Research production of antibody fragments using Immunology 149:533-620, 1998. lower eukaryotic microorganisms. 589-599.

DE GEUS, B. & HENDRIKSEN, C.F.M. In PENNELL, C.A. & ELDIN, P. In vitro vivo and in vitro production of monoclonal production of recombinant antibody antibodies: Current possibilities and future fragments in Pichia Pastoris. 599-603. perspectives. 533-534. LARRICK, J.W., YU, J., CHEN, S., et al. FALKENBERG, F.W. Monoclonal antibody Production of antibodies in transgenic production: Problems and solutions. 542- plants. 603-608. 547.

LIPSKI, L.A., WITZLEB, M.P. & REDDING- ALTSHULER, G.L., DZIEWULSKI, D.M., TON, G.M. Evaluation of small to moder- SOWEK, J.A., et al. (1986). Continuous ate scale in vitro monoclonal antibody hybridoma growth and monoclonal anti- via the use of the i-mabTM gas-permeable body production in hollow fiber reactors- bag system. 547-552. separators. Biotechnology and Bioengineering 28:646-658. PETERSON, N.C. Considerations for in vitro monoclonal antibody production. 553- AMOS, B., AL-RUBEAI, M. & EMERY, A.N. 557. (1994). Hybridoma growth and monoclonal antibody production in a dialysis perfusion MARX, U. Membrane-based cell culture system. Enzyme and Microbial Technology technologies: A scientifically and economi- 16(8):688-695. cally satisfactory alternative to malignant ascites production for monoclonal anti- BLASEY, H.D. & WINZER, U. (1989). Low bodies. 557-559. protein serum-free medium for anti- body production in stirred bioreactors. FALKENBERG, F.W. Production of mono- Biotechnology Letters 11(7):455-460. clonal antibodies in the minipermTM biore- actor: Comparison with other hybridoma BLIEM, R., OAKLEY, R., MATSUOKA, K., et culture methods. 560-570. al. (1990). Antibody production in packed bed reactors using serum-free and protein-free LIPMAN, N.S. & JACKSON, L.R. Hollow medium. Cytotechnology 4(3):279-283. fibre bioreactors: An alternative to murine BOYD, J.E. & JAMES, K. (1989). Human ascites for small scale (<1g) monoclonal Monoclonal Antibodies: Their Potential, antibody production. 571-576. Problems and Prospects. In: Monoclonal SHI, Y., SARDONNI, C.A. & GOFFE, R.A. Antibodies: Production and Application (ed. A. The use of oxygen carriers for increasing Mizrahi). Pp. 1-43. New York NY: Alan R. Liss.

37 BRENNAN, A., DENOMME, L., VELEZ, D., et HEIDEMANN, R., RIESE, U., LUTKEMEYER, al. (1987). A novel perfusion system for D., et al. (1994). The super spinner: A low cost

growth of shear-sensitive hybridoma cells and animal cell culture bioreactor for the CO2 antibody production in a stirred reactor. incubator. Cytotechnology 14(1):1-9. Abstracts from the Annual Meeting of the HEWISH, D. (1996). Ascites and alternatives. American Society of Microbiologists 87:268. Australian and New Zealand Council for the BUGARSKI, B., KING, G.A., JAVANOVIC, G., Care of Animals in Research and Teaching et al. (1989). Performance of an external loop News 9(2):8-10. airlift bioreactor for the production of mono- HOFMANN, F., WRASIDLO, W., DEWINTER, clonal antibodies by immobilized hybridoma D., et al. (1989). Fully Integrated, Compact cells. Applied Microbiology and Biotechnology Membrane Reactor System for the Large 30:264-269.

ccac guidelines Scale Production of Monoclonal Antibodies. FALKENBERG, F.W., HENGELAGE, F.W.T., In: Advances in Animal Cell Biology and KRANE, M., et al. (1993). A simple and Technology for Bioprocesses (eds. R.E. Spier, et al.). 585 pp. Kent, England: Butterworths, inexpensive high density dialysis tubing cell Seven Oaks. culture system for the in vitro production of monoclonal antibodies in high concentration. HOLLEY, M.C. (1992). A simple in vitro Journal of Immunological Methods 165: method for raising monoclonal antibodies 193-206. to cochlear proteins. Tissue Cell 24(5):613- 624. FALKENBERG, F.W., WEICHERT, W.H., KRANE, M., et al. (1995). In vitro production of HOPKINSON, J. (1985). Hollow fiber cell monoclonal antibodies in high concentration in culture systems for economical cell-product a new and easy to handle modular minifer- manufacturing. Bio/Technology 3:225-230. menter. Journal of Immunological Methods INSTITUTE FOR LABORATORY ANIMAL 179:13-29. RESEARCH (ILAR) (1999). Monoclonal GOODALL, M. (1997). Production of mono- Antibody Production: A Report of the clonal antibodies: In vivo versus in vitro Committee on Methods of Producing methods. In: Animal Alternatives, Welfare and Monoclonal Antibodies. ILAR, NRC. Ethics: Proceedings of the 2nd World Washington DC: National Academy Press Congress on Alternatives and Animal Use in (http://grants.nih.gov/grants/policy/ the Life Sciences. (eds. L.F.M. van Zutphen & antibodies.pdf). M. Balls) Pp. 965-972. Amsterdam: Elsevier. JACKSON, L.R. & FOX, J.G. (1995). GORTER, A., VAN DE GRIEND, R.J., VAN Institutional policies and guidelines on EENDENBURG, J.D., et al. (1993). Production adjuvants and antibody production. Institute of bi-specific monoclonal antibodies in a for Laboratory Animal Research Journal 37(3): hollow fibre bioreactor. Journal of Immuno- 141-152. logical Methods 161(2):145-150. JACKSON, L.R., TRUDEL, L.J., FOX, J.G., et al. (1996). Evaluation of hollow fiber biore- HEIDEL, J.R. & STANG, B.V. (1997). actors as an alternative to murine ascites Monoclonal antibody production in gas- production for small scale monoclonal permeable tissue culture bags using serum- antibody production. Journal of Immunological free media. In: CAAT Technical Report Methods 189(2):217-231. #8: Alternatives in Monoclonal Antibody Production, September 1997. JACKSON, L.R., TRUDEL, L.J. & LIPMAN,

38 N.S. (1999). Small-scale monoclonal antibody MARX, U. & MERZ, W. (1995). In Vivo and In production in vitro: Methods and resources. Vitro Production of Monoclonal Antibodies. Lab Animal Autumn special edition:20-30. Bioreactors versus Immune Ascites. In: Methods in Molecular Biology, Vol. 45, JASPERT, R., GESKE, T., TEICHMANN, A., Monoclonal Antibody Protocols (ed. W.C. et al. (1995). Laboratory scale production of Davis). Pp. 169-176. Totowa NJ: Humana monoclonal antibodies in a tumbling chamber. Press. Journal of Immunological Methods 178:77-87. antibody production, 2002 MARX, U., EMBLETON, M.J., FISCHER, R., JENSEN, D'A. & PETRIE SMITH, C. (1999). et al. (1997). Monoclonal antibody production. Selected list of company and institute The report and recommendations of ECVAM resources providing new technologies in Workshop 23. Alternatives to Laboratory monoclonal antibody production. Lab Animal Animals 25(2):121-137 (http://altweb.jhsph. Autumn special edition:31-34. edu/publications/ECVAM/ecvam23.htm).

KAGAN, E., VIERA, E. & PETRIE, H.T. MCARDLE, J. (1997/98). Alternatives to (1997). Comparison of hollow fiber bioreactors ascites production of monoclonal antibodies. and modular minifermentors for the production National Agricultural Library, Beltsville MD. of MABs in vitro. In: CAAT Technical Report Animal Welfare Information Center Newsletter #8: Alternatives in Monoclonal Antibody 8(3-4):1-2, 15-18. Production, September 1997. MORO, A.M., RODRIGUES, M.T.A., KARU, A.E. (1993). Monoclonal Antibodies GOUVEA, M.N., et al. (1994). Multiparametric and Their Use in Detection of Hazardous analyses of hybridoma growth on cylinders in Substances. In: Hazard Assessment of a packed-bed bioreactor system with internal Chemicals-Current Developments (ed. J. areation. Serum-Supplementation and Serum- Saxena). Pp. 205-321. Washington DC: Taylor Free Media Comparison for mAb Production. & Francis International Publishers. Journal of Immunological Methods 176: KUHLMANN, I., KURTH, W. & RUHDEL, I. 67-77. (1989). Monoclonal antibodies: In vitro and in OH, D.J., CHOI, S.K. & CHANG, H.N. (1994). vivo production on a laboratory scale, with a High-density continuous cultures of hybridoma consideration of the legal aspects of animal cells in a depth filter perfusion system. protection. Alternatives to Laboratory Animals Biotechnology and Bioengineering 44(8):895- 17:73-82. 901.

KURKELA, R., FRAUNE, E. & VIHKO, P. PANNELL, R. & MILSTEIN, C. (1992). An (1993). Pilot-scale production of murine oscillating bubble chamber for laboratory scale monoclonal antibodies in agitated, ceramic- production of monoclonal antibodies as an matrix, or hollow-fiber cell culture systems. alternative to ascitic tumours. Journal of Biotechniques 15(4):674-683. Immunological Methods 146:43-48. LERNER, R.A., KANG, A.S., BAIN, J.D., et al. PERSSON, B. & EMBORG, C. (1992). A (1992). Antibodies without immunization. comparison of three different mammalian cell Science 258 (November 20):1313-1314. bioreactors for the production of monoclonal antibodies. Bioprocess Engineering 8(3-4): LOWREY, D.M., MESLOVICH, K., NGUYEN, 157-163. A., et al. (1994). Monoclonal antibody produc- tion and purification using miniature hollow PETRIE, H. (1997). Modular bioreactors as an fiber cell culture technology. American alternative to ascitic fluid production of mono- Biotechnology Laboratory 12(6):16-17. clonal antibodies. Australian and New Zealand

39 Council for the Care of Animals in Research Davis). Totowa NJ: Humana Press. and Teaching News 10(2):6. SHEVITZ, J., REUVENY, S., LAPORTE, T.L., PETROSSIAN, A. & CORTESSIS, G.P. et al. (1989). Stirred tank perfusion reactors (1990). Large-scale production of monoclonal for cell propagation and monoclonal anti- antibodies in defined serum-free media in body production. Advanced Biotechnology airlift bioreactors. Biotechniques 8(4):414-422. Processes 11:81-106. RAMIREZ, O.T., MUTHARASAN, R. & SHUEH, L., SITLANI, S. & BECK, T. (1993). MAGEE, W.E. (1987). A novel immobilized Monoclonal antibody production by artificial hybridoma reactor for the production of mono- capillary hybridoma culture. Joint meeting clonal antibodies. Biotechnology Techniques of the American Association of Immunolo- 1(4):245-250. gists and the Clinical Immunology Society, May 21-25, 1993, Denver CO. Journal of ccac guidelines REUVENY, S. & LAZAR, A. (1989). Immunology 150(8 Pt. 2):327A. Equipment and Procedures for Production of Monoclonal Antibodies in Culture. In: SJÖRGREN-JANSSON, E. & JEANSON, S. Monoclonal Antibodies: Production and (1990). Growing Hybridomas in Dialysis Application (ed. A. Mizrahi). Pp. 45-80. New Tubing, Optimization of Technique. In: Labora- York NY: Alan R. Liss. tory Methods in Immunology (ed. E. Zola). Pp. 1-41. Boca Raton FL: CRC Press. REUVENY, S., VELEZ, D., MACMILLAN, J.D., et al. (1986a). Factors affecting cell growth VAN DER KAMP, M. & DE LEEUW, W. and monoclonal antibody production in stirred (1996). Short review of in vitro methods for reactors. Journal of Immunological Methods monoclonal antibodies. Netherlands Centre for 86:53-59. Alternatives to Animal Use Newsletter 3: 10-12. REUVENY, S., VELEZ, D., MILLER, L., et al. (1986b). Comparison of cell propagation WANG, G.Z., ZHANG, W.Y., FREEDMAN, D., methods for their effect on monoclonal et al. (1992). Use of packed bed reactor antibody yield in fermenters. Journal of for production of monoclonal antibodies. Immunological Methods 86:61-69. Molecular Biology of the Cell 3 (Suppl.):185A. RUHDEL, I. & KUHLMANN, I. (1994). WEICHERT, H., FALKENBERG, F.W., Production of monoclonal antibodies in roller KRANE, M., et al. (1995). In Vitro Production bottles with reduced serum concentrations of Monoclonal Antibodies in High Concentra- and serum substitutes. Bioengineering 10(2): tion in a New and Easy to Handle Modular 15-18, 21-22. Minifermenter. In: Alternative Methods in Toxicology and the Life Sciences Series, Vol. SAXBY, S. (1999). Commercial Production of 11, Proceedings of the World Congress on Monoclonal Antibodies. In: Proceedings of the Alternatives and Animal Use in the Life Production of Monoclonal Antibodies Sciences: Education, Research, Testing (eds. Workshop, August 1999 (eds. J.E. McArdle A.M. Goldberg & L.F.M. van Zutphen). Pp. & C.J. Lund). Alternatives Research and 253-259. New York NY: Mary Ann Liebert, Development Foundation (http://altweb. Inc. jhsph.edu/topics/mabs/ardf/saxby.htm). SCHELLING, M.E. (1995). Methods of WEICHERT, H., KRANE, M., BARTELS, I., Immunization to Enhance the Immune et al. (1994). In vitro production of mono- Response to Specific, Antigens In Vitro. In: clonal antibodies in high concentration in a Methods in Molecular Biology: Vol. 45, new and easy to handle modular minifer- Monoclonal Antibody Protocols (ed. W.C. menter. In Vitro Toxicology 7(2):193.

40